Treatment of CNS tumors with metalloprotease inhibitors

ABSTRACT

The present invention relates to genes and their encoded proteins which regulate neurite growth and the diagnostic and therapeutic use of such proteins (termed herein neurite growth regulatory factors). The proteins of the present invention include central nervous system myelin associated proteins and metalloproteases associated with glioblastoma cells and other malignant tumors which can metastasize to the brain. The metalloproteases of the invention have value in the treatment of nerve damage and of degenerative disorders of the nervous system. The present invention is also directed to inhibitors of the metalloproteases. Such inhibitors in combination with the CNS myelin associated inhibitory proteins can be used in the treatment of malignant tumors.

This patent application is a divisional of application Ser. No.07/401,212, filed Aug. 30, 1989, which is a continuation-in-part of U.S.patent application Ser. No. 07/267,941, filed Nov. 4, 1988, nowabandoned, incorporated by reference herein in its entirety.

TABLE OF CONTENTS

1. Introduction

2. Background of the Invention

2.1. Factors Influencing Neurite Growth

2.2. Proteases and Their Inhibitors

2.3. Neuroblastoma

2.4. Glioblastoma

3. Summary of the Invention

3.1. Definitions

4. Description of the Figures

5. Detailed Description of the Invention

5.1. Isolation and Purification of Neurite Growth Regulatory Factors

5.1.1. Isolation and Purification of CNS Myelin Associated InhibitoryProteins

5.1.2. Isolation and Purification of Receptors for the CNS MyelinAssociated Inhibitory Proteins

5.1.3. Isolation and Purification of Metalloproteases Associated WithMalignant Tumors

5.2. Protein Characterization

5.3. Molecular Cloning of Genes or Gene Fragments Encoding NeuriteGrowth Regulatory Factors

5.3.1. Isolation and Cloning of the Neurite Growth Regulatory FactorGenes

5.3.2. Expression of the Cloned Neurite Growth Regulatory Factor Genes

5.3.3. Identification and Purification of the Expressed Gene Product

5.3.4. Characterization of the Neurite Growth Regulatory Factor Genes

5.4. Production of Antibodies to Neurite Growth Regulatory Factors

5.5. Neurite Growth Regulatory Factor-Related Derivatives, Analogs, andPeptides

5.6. Uses of Neurite Growth Regulatory Factors

5.6.1. Diagnostic Uses

5.6.1.1. CNS Myelin Associated Inhibitory Proteins

5.6.1.2. CNS Myelin Associated Inhibitory Protein Receptors

5.6.1.3. Metalloproteases and their Inhibitors

5.6.2. Therapeutic Uses

5.6.2.1. CNS Myelin Associated Inhibitory Proteins

5.6.2.2. CNS Myelin Associated Inhibitory Protein Receptors

5.6.2.3. Metalloproteases and their Inhibitors

6. Oligodendrocytes and CNS Myelin are Nonpermissive Substrates forNeurite Growth and Fibroblast Spreading in Vitro

6.1. Materials and Methods

6.1.1. Glial Cell Cultures

6.1.2. Glia-Nerve Cell Co-Cultures

6.1.3. Immunofluorescence

6.1.4. Evaluation of Co-Cultures With Nerve Cells, Neuroblastoma Cells,or 3T3 Cells

6.1.5. Preparation of Myelin

6.2. Results

6.2.1. Cultures of Dissociated Young or Adult Rat Optic Nerves

6.2.2. Subtypes of Oligodendrocytes

6.2.3. Response of Various Cell Types to Highly BranchedOligodendrocytes

6.2.3.1. Co-Cultures With Sympathetic or Sensory Neurons

6.2.3.2. Co-Cultures With Fetal Rat Retinal Cells

6.2.3.3. Response Of Other Cell Types To Highly BranchedOligodendrocytes

6.2.4. Absence of Species Specificity

6.2.5. Myelin as a Substrate

6.3. Discussion

7. Two Membrane Protein Fractions From Rat Central Nervous System Myelinwith Inhibitory Properties for Neurite Growth and Fibroblast Spreading

7.1. Materials and Methods

7.1.1. Cell Culture

7.1.2. Sources of Tested Substrates

7.1.3. Substrate Assaying Procedure

7.1.4. Substrate-Processing

7.1.5. Liposomes

7.1.6. Gel-Extracted Protein Fractions As Substrate

7.2. Results

7.2.1. Nonpermissive Substrate Effect is found in CNS Myelin of HigherVertebrates (Chick, Rat), but not Of Lower Vertebrates (Trout, Frog)

7.2.2. Membrane-Bound Protein Fraction of Rat CNS Myelin is Responsiblefor its Nonpermissive Substrate Properties

7.2.3. Identification of 35 kD and 250 kD Minor Proteins from Myelin asNonpermissive Substrates For Fibroblast Spreading and Neurite Outgrowth

7.2.4. Nonpermissive Substrate Property is Enriched in CNS White Matterand in Cultured Oligodendrocytes

7.3. Discussion

8. Antibody Against Myelin-Associated Inhibitor of Neurite GrowthNeutralizes Nonpermissive Substrate Properties of CNS White Matter

8.1. Experimental Procedures

8.1.1. Cell Culture

8.1.2. Substrate Preparation

8.1.3. Immunological Methods

8.1.3.1. Radioimmunoassay

8.1.3.2. Immunoblots

8.1.4. Substrate Testing Procedures

8.1.5. Neurite Growth Into Optic Nerve Explants In Vitro

8.2. Results

8.2.1. Antiserum Against Myelin Neutralizes the Nonpermissive SubstrateEffects of CNS Myelin and of HBOs

8.2.2. IN-1: A Monoclonal Antibody Against Gel Purified 250 kD Inhibitorfrom CNS Myelin Neutralizes Myelin Nonpermissiveness

8.2.3. 250 kD and 35 kD Inhibitors from CNS Myelin Share TwoNeutralizing Epitopes.

8.2.4. IN-1 Specifically Immunoprecipitates Nonpermissive SubstratedActivity from Solubilized Myelin Protein

8.2.5. Nonpermissiveness of Adult Optic Nerve is Neutralized byAbsorption With IN-1 Antibody

8.3. Discussion

9. Involvement of a Metalloprotease in Glioblastoma Infiltration IntoCentral Nervous System Tissue In Vitro

9.1. Materials and Methods

9.1.1. Cell Cultures

9.1.2. Preparation of Nerve Explants for Infiltration Assay

9.1.3. 5CNS Frozen Sections and Myelin as Substrates

9.1.4. C6 Plasma Membranes and Conditioned Medium Preparation

9.1.5. Treatment of CNS Myelin With C6 Plasma Membranes

9.2. Results

9.2.1. C6 Glioblastomas But Not 3T3 Fibroblasts Or B16 MelanomasInfiltrate Optic Nerve and CNS White Matter In Vitro

9.2.2. Glioblastoma Cell Spreading is not Inhibited by CNS Myelin

9.2.3. Specific Blockers of Metalloproteases Inhibit C6 Cell Spreadingon CNS Myelin

9.2.4. A C6 Plasma Membrane-Associated Activity Neutralizes TheInhibitory Substrate Property of CNS Myelin

9.2.5. Inhibitors of Metalloproteases Impair C6 Cell Spreading on CNSWhite Matter and C6 Infiltration of CNS Explants

9.3. Discussion

10. Long Distance Tract Regeneraction in the Lesioned Spinal Cord ofRats by a Monoclonal Antibody Against Myelin-Associated Neurite GrowthInhibitors

10.1. Materials and Methods

10.1.1. Pre-Operative Preparation of Animals, Including Implantation ofHybridoma Cells

10.1.2. Procedure for Performing Spinal Cord Lesion

10.1.3. Post-Lesion Evaluation

10.2. Results: Regeneration of Corticospinal Tract (CST) Fibers OverLong Distances In Rats Bearing IN-1 Secreting Tumors

10.3. Discussion

11. Deposit of Hybridomas

1. INTRODUCTION

The present invention is directed to genes and their encoded proteinswhich regulate neurite growth, antibodies thereto, and the therapeuticand diagnostic uses of such proteins and antibodies. The proteins of thepresent invention include central nervous system myelin associatedinhibitory proteins, and metalloproteases associated with malignanttumors, in particular, primary brain tumors such as glioblastoma andother tumors capable of metastasizing to and spreading in the brain. Thecentral nervous system myelin associated inhibitory proteins inhibitneurite outgrowth and fibroblast spreading and can have important usesin the treatment of malignant tumors. Antibodies to such inhibitoryproteins can have uses in the diagnosis of malignant tumors and in thetreatment of central nervous system damage and degenerative nervediseases. In a specific embodiment of the invention, antibody to neuritegrowth inhibitor may be used to promote the regeneration of neurons overlong distances following spinal cord damage. The metalloproteases of theinvention allow invasive growth of glioblastomas and allow neuriteoutgrowth in central nervous system tissue. They may have important usesin the treatment of central nervous system damage and degenerative nervediseases. Inhibition of the metalloprotease can be therapeuticallyuseful in the treatment of malignant tumors.

2. BACKGROUND OF THE INVENTION 2.1. FACTORS INFLUENCING NEURITE GROWTHIN THE CENTRAL NERVOUS SYSTEM

Cell attachment, cell spreading, cell motility, and, in particular,neurite outgrowth are strongly dependent on cell-substrate interactions(Sanes, 1983, Ann. Rev. Physiol. 45:581-600; Carbonetto et al., 1987, J.Neurosci. 7:610-620). An increasing number of substrate moleculesfavoring neuroblast migration or neurite outgrowth have been found incentral and peripheral nervous tissue (Cornbrooks et al., 1983, Proc.Natl. Acad. Sci. USA 80:3850-3854; Edelman, 1984, Exp. Cell Res.161:1-16; Liesi, 1985, EMBO J. 4:1163-1170; Chiu, A. Y. et al., 1986, J.Cell Biol. 103:1383-1398; Fischer et al., 1986, J. Neurosci. 6:605-612;Lindner et al., 1986, Brain Res. 377:298-304; Mirsky et al., 1986, J.Neurocytol. 15:799-815; Stallcup et al., 1986, J. Neurosci. 5:1090-1101;Carbonetto et al., 1987, J. Neurosci. 7:610-620). The appearance of someof these factors can be correlated with specific developmental stages,and, in the peripheral nervous system (PNS), also with denervation(Edelman, 1984, Exp. Cell Res. 161:1-16; Liesi, 1985, EMBO J.4:1163-1170; Stallcup et al., 1985, J. Neurosci. 5:1090-1101; Daniloffet al., 1986, J. Cell Biol. 103:929-945; Carbonetto et al., 1987, J.Neurosci. 7:610-620). The extracellular matrix protein tenascin has beenshown to possess nonpermissive substrate properties (Chiquet-Ehrismannet al., 1986, Cell 47:131-139).

One of the most characterized of the soluble factors favoring neuriteoutgrowth is nerve growth factor (NGF). NGF promotes nerve fiberoutgrowth from embryonic sensory and sympathetic ganglia in vivo and invitro as well as neurite outgrowth (reviewed in Thoenen et al., 1982,In: Repair and Regeneration of the Nervous System, J. G. Nicholls, ed.,Springer-Verlag, NY, pp. 173-185). NGF may also guide the direction ofsuch neurite outgrowth. Three different molecular forms of NGF have beenrecognized. One type is a dimer (molecular weight ˜26,000) composed oftwo noncovalently linked, identical polypeptide chains. The second formis stable at neutral pH and contains three different polypeptide chains,a, p and 7 (molecular weight ˜140,000). The p chain is the biologicallyactive chain and is identical to the first form of NGF. The third form,which is isolated primarily from mouse L cells, (see U.S. Pat. No.4,230,691, by Young, issued Oct. 28, 1980, and references therein) has amolecular weight of about 160,000 but is unstable at neutral pH. NGF hasthus far been isolated from the submandibullar glands of mice, mouse Lcells, and the prostate gland of the guinea pig and bull (reviewed inThoenen et al., 1982, supra). No differences between the biologicalaction of mouse, guinea pig and bull NGF have been detected. Inaddition, NGF isolated from mice have been found to bind to the humanNGF receptor (Johnson et al., 1986, Cell 47:545-554).

The differentiated central nervous system (CNS) of higher vertebrates iscapable of only very limited regenerative neurite growth after lesions.Limited regeneration after lesion has been seen in the retina (McConnelland Berry, 1982, Brain Res. 241:362-365) and in aminergic unmyelinatedfiber tracts after chemical (Bjorklund and Stenevi, 1979, Physiol. Rev.59:62-95) but not mechanical lesions (Bregman, 1987, Dev. Brain Res.34:265-279). Neurite growth from implanted embryonic CNS tissues inadult rat CNS has been found in some cases to reach up to 14 mm withinsome gray matter areas, but has not been found to exceed 1 mm withinwhite matter (Nornes et al., 1983, Cell Tissue Res. 230:15-35; Bjorklundand Stenevi, 1979, Physiol. Rev. 59:62-95; Commission, 1984,Neuroscience 12:839-853). On the other hand, extensive regenerativegrowth has been found in the CNS of lower vertebrates and in theperipheral nervous system of all vertebrates including man. Results fromtransplantation experiments indicate that the lack of regeneration isnot an intrinsic property of CNS neurons, as these readily extendprocesses into implanted peripheral nervous tissue (Benfey and Aguayo,1982, Nature (London) 296:150-152; Richardson et al., 1984, J.Neurocytol. 13:165-182 and So and Aguayo, 1985, Brain Res. 328:349-354).PNS neurons, however, failed to extend processes into CNS tissue, thusindicating the existence of fundamental differences between the twotissues (Aguayo et al., 1978, Neurosci. Lett. 9:97-104; Weinberg andSpencer, 1979, Brain Res. 162:273-279).

One major difference between PNS and CNS tissue is the differentialdistribution of the neurite outgrowth promoting extracellular matrixcomponent laminin (Liesi, 1985, EMBO J. 4:2505-2511; Carbonetto et al.,1987, J. Neurosci. 7:610-620). Other factors though may be involved.Drastic differences have been observed in neurite growth supportingproperties of sciatic and of optic nerve explants in vitro, in spite ofthe presence of laminin immunoreactivity in both explants (Schwab andThoenen, 1985, J. Neurosci. 5:2415-2423). These experiments were carriedout in the presence of optimal amounts of neurotrophic factors anddifferences persisted upon freezing of tested substrates.

It has been suggested that the differentiated CNS may lack cellular orsubstrate constituents that are conducive for neurite growth duringdevelopment (Liesi, 1985, EMBO J. 4:2505-2511; and Carbonetto et al,1987, J. Neurosci. 7:610-620), or it may contain components which arenonpermissive or inhibitory for nerve fiber regeneration (Schwab andThoenen, 1985, J. Neurosci. 5:2415-2423).

Recently, a growth (cell proliferation) inhibitory factor for mouseneuroblastoma cells was partially purified and characterized from theculture medium of fetal rat glioblasts as well as from C6 rat gliomacells (Sakazaki et al., 1983, Brain Res. 262:125-135). The factor wasestimated to have a molecular weight of about 75,000 by gel filtrationwith BioGel P-20 with an isoelectric point of 5.8. The factor did notappear to alter the growth rate or morphology of glial cells (C6) orfibroblasts (3T3). In addition, no significant nerve growth inhibitoryfactor activity was detected towards neuroblastoma cells (Neuro La,NS-20Y and NIE-115) or cloned fibroblasts (3T3).

2.2. PROTEASES AND THEIR INHIBITORS

Different proteolytic activities have in the past been shown to beincreased in tumorigenic cell lines (Matrisian et al., 1986, Proc. Natl.Acad. Sci. U.S.A. 83:9413-9417; Mignatti et al., 1986, Cell 47:487-498),in primary tumor explants (Mullins and Rohrlich, 1983, Biochem. Biophys.Acta 695:177-214), or in transformed cells (Quigley, 1976, J. Cell Biol.71:472-486; Mahdavi and Hynes, 1979, Biochem. Biophys. Acta 583:167-178;Chen et al., 1984, J. Cell Biol. 98:1546-1555; Wilhelm et al., 1987,Proc. Natl. Acad. Sci. U.S.A. 84: 6725-6729). One such group ofproteases, metalloproteases has been shown to be involved in a number ofmembrane events, including myoblast fusion (Couch and Stritmatter, 1983,Cell 32:256-265), and exocytosis in mast cells (Mundy and Stritmatter,1985, Cell 40:645-656).

The isolation and characterization of a plasma membrane-boundmetalloprotease (endopeptidase 24.11, enkephalinase) was reported byAlmenoff and Orlowski (1983, Biochemistry 22:590-599). A metalloproteaseexpressed by Rous sarcoma virus transformed chick embryo fibroblastswhich degrades fibronectin and which was localized at adhesion sites andon "invadopodia" was described by Chen and Chen (1987, Cell 48:193-203).

Studies indicate that proteases and their inhibitors can influenceneurite extension in neuroblastoma cells (Monard et al., 1983, Prog.Brain Res. 58:359-363) and in cultured neonatal mouse sensory ganglia(Hawkins and Seeds, 1986, Brain Res. 398:63-70). Cultured glial cellsand gliomas were found to release a 43 kD protein, a glia derivedneurite promoting factor (GdNPF), which induces neurite outgrowth inneuroblastoma cells but inhibits cell migration (Monard, et al., 1983,supra). GdNPF was shown to be a very potent inhibitor of cell surfaceassociated serine protease activity. Neurite outgrowth from normal mousesensory ganglia can be enhanced by the addition of serine proteaseinhibitors, ovomucoid trypsin inhibitor, leupeptin, soybean trypsininhibitor, or thrombin (Hawkins and Seeds, 1986, supra). In contrast,proteases were found to inhibit such neurite outgrowth. Results frompreliminary studies indicate that such proteases possess a thrombin ortrypsin like activity (Hawkins and Seeds, 1986, supra).

Other proteases have also been characterized though their functionalrole in neurite outgrowth is as yet unknown. These include aurokinase-like plasminogen activator and a calcium dependentmetalloprotease released by sympathetic and sensory rat neurons(Pittman, 1985, Dev. Biol. 110:911-101). The metalloprotease was foundto have a molecular weight of 62 kD, to require 1 mM Ca²⁺ for calciumactivity, and to degrade native and denatured collagen more readily thancasein, albumin, or fibronectin. The plasminogen activator was found tohave a molecular weight of 51 kD, and was precipitated by a rabbitantiserum produced against human urokinase. It may be converted to itsactive form of 32 kD.

2.3. NEUROBLASTOMA

Neuroblastoma arises from neuroectoderm and contains anaplasticsympathetic ganglion cells (reviewed in Pinkel and Howarth, 1985, In:Medical Oncology, Calabrese, P., Rosenberg, S. A., and Schein, P. S.,eds., MacMillan, N.Y., pp. 1226-1257). One interesting aspect ofneuroblastoma is that it has one of the highest rates of spontaneousregression among human tumors (Everson, 1964, Ann. NY Acad. Sci.114:721-735) and a correlation exists between such regression andmaturation of benign ganglioneuroma (Bolande, 1977, Am. J. Dis. Child.122:12-14). Neuroblastoma cells have been found to retain the capacityfor morphological maturation in culture. The tumors may occur anywherealong the sympathetic chain, with 50% of such tumors originating in theadrenal medulla.

Neuroblastoma affects predominantly preschool aged children and is themost common extracranial solid tumor in childhood, constituting 6.5% ofpediatric neoplasms. One half are less than two years of age upondiagnosis. Metastases are evident in 60% of the patients at presentationusually involving the bones, bone marrow, liver, or skin. The presentingsymptoms may be related to the primary tumor (spinal coral compression,abdominal mass), metastatic tumor (bone pain) or metabolic effects ofsubstances such as catecholamines or vasoactive polypeptides secreted bythe tumor (e.g. hypertension, diarrhea).

Experimental evidence indicates that an altered response to NGF isassociated with neuroblastoma (Sonnenfeld and Ishii, 1982, J. Neurosci.Res. 8:375-391). NGF stimulated neurite outgrowth in one-half of theneuroblastoma cell lines tested; the other half was insensitive.However, NGF neither reduced the growth rate nor enhanced survival inany neuroblastoma cell line.

Present therapies for neuroblastoma involve surgery and/or chemotherapy.Radiation therapy is used for incomplete tumor responses tochemotherapy. There is a 70-100% survival rate in individuals withlocalized tumors, but only a 20% survival rate in those with metastaticdisease even with multiagent chemotherapy. It appears that patients lessthan one year have a better prognosis (70%) than older children.

2.4. GLIOBLASTOMA

Glioblastoma is a highly malignant astrocytic tumor usually located inthe cerebral hemisphere. Astrocytes appear to be a supporting tissue forneurons and comprise the vast majority of the intraparenchymal cells ofthe brain (reviewed in Cutler, 1987, In: Scientific American Medicine V.2, Rubenstein and Federman, eds., Scientific American, Inc., NY, pp.1-7). Results from a survey conducted by the National Institute ofNeurological and Communicative Disorders and Stroke indicated that theincidence of primary brain tumors in the United States is approximatelyeight per 100,000, in which 20% of those tumors are glioblastomas. Thesetumors are generally found in individuals between 45 and 55 years ofage. The tumors may also involve multiple lobes and may rupture into theventricular system or extend across the corpus collosum to the oppositehemisphere. Due to the resulting increase in intracranial pressure,symptoms of tumor growth include headache, nausea and vomiting, mentalstatus changes, and disturbances of consciousness. Due to their highlyinvasive properties, glioblastomas are associated with a poor prognosis.Chemotherapeutic agents or radiotherapies may be used. However, patientsgenerally do not survive longer than two years even with thesetherapies.

3. SUMMARY OF THE INVENTION

The present invention relates to genes and their encoded proteins whichregulate neurite growth and the diagnostic and therapeutic uses of suchproteins. Such proteins are termed herein neurite growth regulatoryfactors. The neurite growth regulatory factors of the present inventioninclude, in one embodiment, central nervous system myelin associatedproteins which inhibit neurite outgrowth, and are termed herein neuritegrowth inhibitory factors. Another embodiment of the invention isdirected to neurite growth regulatory factors which are metalloproteasesassociated with malignant tumors, in particular, those tumors metastaticto the brain. Such metalloproteases enable the malignant cells toovercome the inhibitory CNS environment and invade large areas of brainand spinal cord.

The CNS myelin associated proteins inhibit neurite outgrowth in nervecells and neuroblastoma cells and also inhibit the spreading offibroblasts and melanoma cells. Such inhibitory proteins include but arenot limited to 35,000 dalton and a 250,000 dalton molecular weightproteins and analogs, derivatives, and fragments thereof. The CNS myelinassociated inhibitory proteins may be used in the treatment of patientswith malignant tumors which include but are not limited to melanoma andnerve tissue tumors (e.g., neuroblastoma). The absence of the myelinassociated inhibitory proteins can be diagnostic for the presence of amalignant tumor such as those metastatic to the brain (e.g.,glioblastoma). The present invention also relates to antagonists of theCNS myelin associated inhibitory proteins, including, but not limitedto, antibodies, i.e. antibodies IN-1 or IN-2. Such antibodies can beused to neutralize the neurite growth inhibitory factors forregenerative repair after trauma, degeneration, or inflammation. In afurther specific embodiment, monoclonal antibody IN-1 may be used topromote regeneration of nerve fibers over long distances followingspinal cord damage.

The present invention further relates to neurite growth regulatoryfactor receptors and fragments thereof as well as the nucleic acidsequences coding for such neurite growth regulatory factor receptors andfragments, and their therapeutic and diagnostic uses. Substances whichfunction as either agonists or antagonists to neurite growth regulatoryfactor receptors are also envisioned and within the scope of the presentinvention.

The metalloproteases of the present invention can be found associatedwith malignant tumors, in particular, those capable of metastasizing tothe brain. In a specific embodiment, the metalloprotease is associatedwith membranes of glioblastoma cells. The metalloproteases, and analogs,derivatives, and fragments thereof can have value in the treatment ofnerve damage resulting from trauma, stroke, degenerative disorders ofthe central nervous system, etc. In another embodiment of the invention,the metalloprotease may be used in combination with antibodies to theneurite growth inhibitory factors to treat nerve damage.

The present invention is also directed to inhibitors of and/orantibodies to the metalloproteases of the invention. Such inhibitorsand/or antibodies can be used in the diagnosis and/or treatment ofmalignant tumors such as those which can metastasize to the brain,including but not limited to glioblastomas. Alternatively, themetalloprotease inhibitors, in combination with CNS myelin associatedinhibitory protein or analogs, derivatives, or fragments thereof, may beused in the treatment and/or diagnosis of malignant tumors including butnot limited to glioblastoma, neuroblastoma, and melanoma.

3.1. DEFINITIONS

As used herein, the following terms shall have the meanings indicated:

BSA: bovine serum albumin

cbz-tyr-tyr: carbobenzoxy-tryosine-tyrosine

cbz-gly-phe-NH₂ : carbobenzoxy-glycine-phenylalanine-amide

cbz-ala-phe-NH₂ : carbobenzoxy-alanine-phenylalanine-amide

cbz-phe-phe-NH₂ : carbobenzoxy-phenylalanine-phenylalanine-amide

cbz-gly-phe-phe-NH₂ :carbobenzoxy-glycine-phenylalanine-phenylalanine-amide

CNS: central nervous system

CST: Corticospinal tract

DMEM: Dulbecco's Modified Minimal Essential Media

EDTA: ethylenediamine tetracetate

EGTA: ethylene glycol-bis-(β-aminoethyl ether)-N,N,N'-N'-tetracetate

FCS: fetal calf serum

FITC: fluorescein isothiocyanate

GdNPF: glial-derived neurite promoting factor

GFAP: glial fibrillary acid protein

HBO: highly branched oligodendrocyte

Hepes: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid

IN-1: a monoclonal antibody against gel-purified 250 kD CNS myelinassociated inhibitory protein

IN-2: a monclonal antibody against gel-purified 35 kD CNS myelinassociated inhibitory protein

J1: a cell adhesion molecule of molecular weight 160-180 kD

kD: kilodalton

Mab: monoclonal antibody

MW: molecular weight

N-CAM: neural cell adhesion molecule

NGF: nerve growth factor

neurite growth

regulatory factors: CNS myelin associated 35 kD and 250 kD inhibitoryproteins, and a glioblastoma cell membrane associated metalloprotease

PBS: phosphate buffered saline

PLYS: poly-D-lysine

PNS: peripheral nervous system

PORN: polyornithine

SCG: superior cervical ganglion

SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Tris: Tris (hydroxymethyl) aminomethane

4. DESCRIPTION OF THE FIGURES

FIGS. 1A-H. Sympathetic (A-D) or retinal (E) neurons plated intocultures of optic nerve glial cells show nonpermissive substrate effectof highly branched oligodendrocytes and the absence of such effect inimmature oligodendrocytes. FIGS. 1A, C, E, G show phase contrastpictures. FIGS. 1B, D, F show immunofluorescence with antibody O₄. FIG.1H shows immunofluorescence with antibody O₁.

FIGS. 1A and 1B show "windows" (areas free of neurites)formed by highlybranched oligodendrocytes (10-day-old optic nerves, 18 days in vitro) inthe neurite plexus of sympathetic neurons (A: 8 days in vitro; B: 4 daysin vitro). Magnification: ×120. In FIG. 1B, a neurite changing itsdirection is seen (arrow-head). Schwann cells also avoid theoligodendrocyte.

FIGS. 1C and 1D show antibody O₄ -positive oligodendrocytes (from10-day-old optic nerves, 7 days in vitro) surrounded by plexus ofsympathetic neurites (5 days in vitro). Magnification: ×220. Neuritescharacteristically "loop around" the oligodendrocytes. The occasionalspanning of neurite bundles over nonpermissive oligodendrocytes occursas a secondary event.

FIGS. 1E and 1F show that antibody O₄ -positive cells with the typicalmorphology of immature oligodendrocytes are permissive for sympatheticneurites (arrow-heads) (5 days in vitro). Magnification: ×380.

FIGS. 1G and 1H show that E 20 rat retinal cells (2 days in vitro) donot adhere or grow neurites onto highly branched, antibody O₁ -positiveoligodendrocytes. Magnification: ×200.

FIGS. 2A-D. A, B: Histograms showing the frequency ofinteractions/overlap of sympathetic neurites and Schwann cells withhighly branched (A) or immature (B) oligodendrocytes. Glial cells from 8to 10-day optic nerves (2 days in vitro) were co-cultured for anadditional 2 days with dissociated neurons from superior cervicalganglia and then stained with antibody O₄. Oligodendrocytes wereclassified by morphology on coded fluorescence pictures. On phasecontrast pictures, the fractional area of contact with neurites andSchwann cells was determined and classified into 3 categories: <20%, 20to 80%, or >80% of oligodendrocyte territory covered by neurites orSchwann cells. Values represent mean frequencies of cells in 3categories±SEM (standard error of the mean) (4 cultures; 70 to 130systematically sampled cells per culture).

C, D: Histograms showing the interaction of retinal cells with highlybranched (C) or immature (D) oligodendrocytes. Glial cultures from adultrat optic nerves (6-11 days in vitro) were co-cultured for 1-5 days withembryonic rat retinal cells. Antibody O₄ stained oligodendrocytes wereclassified morphologically, and the total area occupied by eacholigodendrocyte as well as the fraction occupied by retinal cells wasdetermined by measuring with a graphic tablet. n=109.

FIGS. 3A-D. Astrocytes represent an adhesive substrate for neurons andneurites.

FIGS. 3A and 3B show sympathetic neurites (13 days in vitro) growing onreactive protoplasmic astrocyte (arrow in A); GFAP POSITIVE (B): from10-day-old rat optic nerve, 23 days in vitro. Magnification: ×220.

FIGS. 3C and D show retinal cells (from E17 retina, 2 days in vitro)adhering to astrocytes (GFAP-positive, FIG. 3D); from 10-day-old opticnerve, 9 days in vitro) with long and with short (arrow) processes.Magnification: ×400.

FIGS. 4A-F. A, B: Highly branched oligodendrocytes (O₄ -positive) arenon-permissive for attachment and fiber outgrowth of NB-2A neuroblastomacells. NB-2A cells were cultured for 24 hours on optic nerve glial cells(6-day old rat optic nerves, 3 days in culture) and stimulated forneurite outgrowth by GdNPF (Guenther et al., 1985, EMBO J. 4:1963-1966).NB-2A cells adjacent to oligodendrocytes (short arrows) show assymmetricougrowth; distant cells (long arrows) show random orientation ofoutgrowth. Magnification: ×260. C-F: 3T3 fibroblasts plated at high celldensities into optic nerve glial cultures show nonpermissive substrateeffect of highly branched oligodendrocytes (C, E). The oligodendrocytein C/D has large membrane areas connecting its process network. Animmature oligodendrocyte (E, F: arrow; O₄ -positive, irregularmorphology) is overgrown by spreading fibroblasts. 10-(C, D) and 12-(E,F) day old optic nerves, 2 days int, vitro; 3T3 added for 3 hours. D, F:O₄ -staining. C, D: magnification is ×300; E, F: magnification is ×250.

FIG. 5. Orientation of neuroblastoma process outgrowth in relation tohighly branched oligodendrocytes. Optic nerve glial cells (2 or 6 daysin vitro) were co-cultured with NB-2A cells for 24 hours in presence ofGdNPF or dibutyryl CAMP. Antibody O₄ -positive highly branchedoligodendrocytes were systematically sampled and neighbouringneuroblastoma cells were classified as adjacent when the distancebetween the edge of oligodendrocyte process network and neuroblastomacell body was less than 2 cell body diameters. Neuroblastoma cells atgreater distances were classified as distant. Neuroblastoma processeswere assigned to four sectors (1-4) according to their direction withregard to the closest oligodendrocyte as illustrated. Values, shown inTable IA (Section 6.2.3.3., infra) represent means±SEM of 3 experiments(60-100 neurites from 3 cultures per experiment). * p<0.05; * * *p<0.001.

FIGS. 6A-B. Histograms showing the overlap of 3T3 cells with highlybranched (A) or immature (B) oligodendrocytes. 3T3 cells wereco-cultured for 3-4 hours on optic nerve glial cells at high celldensity, and cultures fixed and stained with antibody O₄.Oligodendrocytes were sampled systematically, classified as highlybranched or immature oligodendrocytes, and their overlap with 3T3 cellswas determined in the 3 categories indicated. Values represent means±SEM(standard error measurement) of four experiments (70-170 cells).

FIGS. 7A-D. Inhibition of neurite outgrowth by use of CNS myelin as asubstrate. Sympathetic neurons (from 1-day-old rat superior cervicalganglia) cultured in the presence of 100 ng/ml NGF for 26 hours onpoly-D-lysine (PLYS)-coated culture dish containing focal spots of CNSor PNS myelin. CNS myelin (A, C) strongly inhibits neurite outgrowth;PNS myelin (B, D) is a permissive substrate. In C and D, the border of amyelin islet on the PLYS is shown. Magnification: ×75.

FIGS. 8A-B. Nonpermissive substrate effects of CNS myelin but not PNSmyelin for neurite outgrowth from neuroblastoma cells (A) and for 3T3cell spreading (B).

FIG. 8A shows neuroblastoma cells cultured for 5 hours in the presenceof 2 mM dibutyrylcyclic AMP on PLYS (solid bars), CNS myelin coated PLYS(hatched bars), or PNS myelin-coated PLYS (open bars). Cells wereclassified as round cells, filopodia or short process carrying cells, orcells with processes longer than 1 cell diameter. Values representmeans±SEM of 3 cultures (250-450 cells per culture).

FIG. 8B shows 3T3 cells cultured for 1 hour on PLYS, CNS myelin-coatedPLYS, or PNS myelin-coated PLYS. Cells were classified as round cells,cells with filopodia or short processes, or large flat cells. Valuesrepresent means±SEM of 3 cultures (300-400 cells per culture).

FIG. 9. Substrate properties of CNS myelin fractions from rat, chick,trout, and frog. Spinal cord myelin fractions from different specieswere adsorbed to PLYS-coated wells of dishes (Greiner). 3T3 cells wereadded and experiments were scored after 1 hour. Spreading values aregiven as mean±SEM. Substrates: myelin fractions from: (1) rat CNS; (2)chick CNS; (3) trout CNS; (4) frog CNS; (5) rat PNS.

FIG. 10. SDS-PAGE fractionation of rat CNS myelin protein. Nonpermissivesubstrate proteins comigrates with proteins of 250 and of 35 kD on a3-15% polyacrylamide-reducing gradient gel of rat CNS myelin protein.Protein from the indicated gel regions was extracted andactivity-containing regions of ˜35 and 250 kD proteins were determinedby assaying the nonpermissiveness of corresponding liposomes. Molecularmasses were estimated from commercial standards (Sigma Chemical Co.)

FIGS 11A-C. 35 and 250 kD (11A and 11B, respectively) protein fractionsfrom rat CNS myelin are nonpermissive substrates for 3T3 spreading andneurite outgrowth. Liposomes formed in the presence of gel-extractedprotein fractions as indicated were tested for their substrateproperties. Substrate designated as rest: protein from pooled gelregions excluding the 35 kD and 250 kD fractions. Incubation times wereone (3T3 cells) and 24 hours (SCG neurons in the presence of NGF),respectively. Bars: (3T3) 100 μm; (SCG) 50 μm.

FIG. 12. 35 kD and 250 kD protein fractions from rat CNS myelin convertpermissive substrates into nonpermissive substrates. Total protein (T)liposomes were formed with 100 μg of protein from each of the threesources (rat) indicated in the figure. (1) (250 kD) and (2) (35 kD)liposomes were formed with gel-extracted protein regions from 3-15% gelsloaded with 500 μg protein from the same three sources. Columns labeled+1 and +2 indicate that 250 kD (1) or 35 kD (2) protein from 500 μg ofrat CNS myelin were combined with 100 μg of total liver or PNS myelinprotein before reconstitution.

FIGS. 13A-H. Nonpermissive substrate properties of CNS myelin and of 35kD and 250 kD inhibitors were neutralized by monoclonal antibody IN-1.SCG neurons were cultivated on test substrates in the absence (A, C, E,and G) or the presence (B, D, F, and H) of monoclonal antibody IN-1.Cultures were photographed after 24 hours. Substrate-adsorbed wells ofGreiner dishes were preincubated in the presence of hybridoma medium orIN-1 hybridoma supernatant for 30 minutes, four-fifths of thepreincubation medium was then removed, and SCG neurons were added, thusreplacing the removed quantity of medium. Well-adsorbed substrates wereas follows: CNS myelin membranes (A and B), 250 kD inhibitor-containingliposomes (C and D), 35 kd inhibitor-containing liposomes (E and F), andliposomes containing permissive 250 kD protein fraction from rat PNSmyelin (G and H). Bar, 50 μm.

FIGS. 14A-F. Monoclonal antibody IN-1 binds to the surface of HBOs andneutralizes their nonpermissive substrate properties. Two day old opticnerve cultures were either stained with IN-1 (B) or tested as substratesfor 3T3 cell spreading in the presence (C,D) and absence (E,F) of IN-1.IN-1 specifically bound to the surface of HBOs (A, phase contrast; B,immunofluorescence with antibody IN-1). Frequent fibroblast spreadingover HBO territories was observed when IN-1 was present in theincubation medium (C, phase contrast; D, immunofluorescence with O₁ ;cells were fixed 2 hours after the addition of 3T3 fibroblasts withfixation media containing 4% paraformaldehyde before incubation withantibody O₁), whereas fibroblasts did not invade the territory of HOs inthe presence of control monoclonal antibody O₁ (E, phase contrast; F,immunofluorescence with O₁). In the control experiment of (E), cellswere fixed 10 hours after fibroblast addition to show attachment of HBOprocesses to 3T3 cells; numerous O₁ ⁺ processes were observed connectingoligodendrocyte and fibroblast cell bodies. Bar, 50 μm.

FIG. 15. Quantitative determination of IN-1-mediated neutralization ofHBO nonpermissive substrate properties. 3T3 cells were added to 2 dayold optic nerve cultures in the presence of either hybridoma medium (C),monoclonal antibody O₄ or antibody IN-1. Cultures were fixed after 2hours, and oligodendrocytes were identified by O₁ staining. Preferentialadhesion and spreading of 3T3 cells on a polylysine-coated culture dishand on the surface of O₁ ⁺ HBOs were determined as-described infra inSection 8.1.4. A value of 100% inhibition represents no overlap of 3T3cell surfaces with O₁ ⁺ surfaces; 0% inhibition represents no apparentdiscrimination by 3T3 cells between the polylysine-coated culture dishand HBO surface; negative inhibition values indicate that the fractionof HBO surfaces covered by 3T3 cells was larger than the fraction of theentire culture surface covered by 3T3 cells, i.e., 3T3 cellspreferentially spread on HBOs. Values are means±SEM. Twenty separatedeterminations from two independent experiments were analyzed.

FIG. 16. Immunoblot of rat CNS myelin protein with monoclonal antibodyIN-1. CNS myelin protein was separated by 3-15% SDS-PAGE under reducingconditions. Lane 1, silver-stained gel of rat CNS myelin protein; thepositions of the inhibitory protein regions are indicated by arrowheads.Lanes 2 and 3, immunoblot with IN-1 (lane 2) or control antibody againsthorseradish peroxidase (lane 3). Lane 1 and lanes 2 and 3 are from twodifferent gels. The approximate migration position of protein bandsgiving specific IN-1⁺ signals are indicated by arrowheads (lanes 1 and2). IN-1 binding to protein bands in the 35 kd region was variable,weak, and not detectable on the immunoblot shown.

FIGS. 17A-B. Laminin immunoreactivity in adult optic nerve in vivo andin cultured optic nerve explants. In the in vivo nerve, only subpial andperivascular basement membranes showed specific laminin immunoreactivity(A). In cultured optic nerve explants (chamber culture, 4 weeks invitro), strongly laminin-positive cells, presumably astrocytes (arrows),appeared inside the explant (B). Bar, 50 μm.

FIGS. 18A-B. Sensory axons in IN-1-injected nerve explants. Electronmicrographs from representative experiments as described in Table X. (A)Electron micrograph of IN-1-injected optic nerve 1 mm from the proximalstump; an axon bundle growing in direct contact with the myelin isshown. (B) In the presence of IN-1, numerous axons grew 3 mm into theoptic nerve explant. Bar, 0.5 μm.

FIGS. 19A-D. C6, but not 3T3 cells, infiltrate optic nerve explants.Phase-contrast microphotographs of μm frozen sections of rat optic (A,B)or sciatic (C,D) nerve explants, after 2 weeks incubation with C6 (A,C)or 3T3 (B,D) cells. Cells were added to one tip of the explants.Infiltrated cells can be seen after cresyl violet staining in bothsciatic nerves (C,D) but only for C6 cells in the optic nerve (A).Arrows in (B) point to few 3T3 cells adjacent to blood vessels. Bar, 0.2mm.

FIGS. 20A-F. C6, but not 3T3 or B16 cells attach and spread on CNS whitematter of rat cerebellar frozen sections. Phase contrast micrographs ofrat cerebellar frozen sections (25 μm) on which C6 (a,b), 3T3 (c,d) orB16 (e) cells were cultured for 2 days. A clear difference on whitematter (wm) emerges for 3T3 and B16 cells compared to C6 cells. gl:granular layer, ml: molecular layer. Gray matter is composed of granularand molecular layer. Bar, 0.3 mm.

FIGS. 21A-C. C6 cells overcome the inhibitory substrate property of CNSmyelin. Spreading of C6 (A), 3T3 (B) and B16 (C) cells on PLYS or CNSmyelin. Spreading is calculated as described in Section 9.1.3., infrausing electron micrographs. 0% spreading: round cells; 100% spreadingwas taken as the average value at 300 minutes.

FIG. 22. 1,10-phenanthroline inhibits C6 spreading specifically on CNSmyelin. C6 cells were cultured for 3 hours on the indicated substratesin the presence of increasing doses of 1,10-phenanthroline. Spreadingwas inhibited by low doses exclusively on CNS myelin.1,10-phenanthroline concentrations above 0.5 mM exert a general toxiceffect on all substrates. Spreading was quantified as indicated in FIG.21.

FIGS. 23A-D. Degradation of CNS inhibitory substrate by C6 plasmamembranes is 1,10-phenanthroline sensitive. Spreading of 3T3 cells onCNS myelin was induced by pretreatment of myelin with C6 plasmamembranes. 1,10-phenanthroline abolished this effect. Shown are phasecontrast micrographs of 3T3 cells on polylysine (PLYS) (23A), on CNSmyelin (23B), on CNS myelin pretreated with C6 plasma membranes, and onCNS myelin pretreated with C6 plasma membranes (C6-PM) (23C) in thepresence of 1,10-phenanthroline (23D).

FIGS. 24A-B. C6 cell attachment and spreading on CNS white matter of ratcerebellar frozen section is impaired by metalloprotease blockers. Phasecontrast micrographs of C6 cells on rat cerebellar frozen sectionscultivated in the presence of either 0.1 mM cbz-ala-phe (a) or 0.1 mMcbz-tyr-tyr (b). Inhibition of attachment and spreading is particularlyevident in the center of the white matter (asterisks), but is alsovisible in the main white matter branches (arrows).

FIG. 25. C6 cell infiltration into CNS explants is impaired bycbz-tyr-tyr. Cells were added to one tip of optic nerve explants(chamber cultures) in the presence of the metalloprotease inhibitorcbz-tyr-tyr, or of the control peptide cbz-ala-phe 14 day old cultureswere quantified. Infiltrated cells were counted in the first 1.3 mm ofthe explants. Each column represents the number of infiltrated cells per0.1 mm. Only the most central part of the explants was considered (0.25mm). Values represent means±SEM of two sets of experiments for a totalof 8 explants.

FIGS. 26A-B. Tumor of antibody-secreting mouse IN-1 hybridoma cells inthe cortex of an 8 day old rat. 1 mio. cells (in 2 μl) were injected atP2; (A) Cresyl violet stained frozen section (15 μl) shows wellcircumscribed, compact tumor (arrow); (B) Antibody productiondemonstrated by immunofluorescence with anti-mouse-Ig-FITC. Tumor andsurrounding tissue up to the lateral ventricle (small arrow) arestrongly stained (adjacent section to A). Rat was fixed by perfusionwith 4% formaldehyde in phosphate buffer. Magnification: 6.4×

FIG. 27. Labelled corticospinal tract fibers are present far distal tospinal cord lesions in 4 IN-1 treated rats (top), but not in 4 controlanti-HRP treated animals (bottom). Camera lucida drawings oflongitudinal sections of spinal cords (75μ total thickness, 3superimposed 25μ sections) showing labelling pattern of CST fiberslabelled by anterograde transport of WGA-HRP. Long-distance elongationof regeneration CST fibers is present in the IN-1 treated rats. Arrowpoints to lesion sites, which sometimes contain small cavernscommunicating with the central canal.--r: rostral; c: caudal.

FIG. 28. Dark-field micrographs of spinal cord longitudinal sections ofan IN-treated rat (A-D) and an anti-HRP-treated rat (E-G). highmagnification micrographs show the densely labelled CST with a broadsprouting zone rostral to the lesion (B and F). Fine grain specificlabel (arrows) is seen immediately caudal to the lesion in both animals(B, F). Label is also present far distal (about 7 mm) in the IN-treatedspinal cord (C,D). In contrast, no such label was detectable in theanti-HRP-treated rat (G).

Large white dots (crystals) in all sections represent reaction productin blood vessels, always prominant in material reated with this highlysensitive procedure. c=caudal; L=lesion; r=rostral.

Magnification: A,E: 14×; B,F,G: 70×; C,D: 140×.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to genes and their encoded proteinswhich regulate neurite growth and the diagnostic and therapeutic uses ofsuch proteins. The proteins of the present invention (termed hereinneurite growth regulatory factors) include proteins associated withcentral nervous system myelin with highly nonpermissive substrateproperties, termed herein neurite growth inhibitory factors. The neuritegrowth regulatory factors also include metalloproteases which can befound associated with malignant tumors, in particular, those tumorsmetastatic to the brain.

The CNS myelin associated proteins of the invention inhibit neuriteoutgrowth in nerve cells or neuroblastoma cells. The protein can alsoinhibit fibroblast spreading and migration. These inhibitory proteinsare active cross-species and may be used in the treatment of patientswith malignant tumors including but not limited to melanoma and tumorsof nerve tissue (e.g. neuroblastoma). In a specific example of thepresent invention, a 35 kilodalton and a 250 kilodalton CNS myelinassociated protein are described.

The present invention is also directed to antibodies to the CNS myelinassociated proteins and their therapeutic and diagnostic uses. Theseantibodies can be used in the treatment of nerve damage resulting from,e.g., trauma (e.g., spinal cord injuries), stroke, degenerativedisorders of the central nervous system, etc. In particular, antibodiesto CNS myelin associated proteins may be used to promote regeneration ofnerve fibers. In a specific embodiment of the invention, monoclonalantibody IN-1 may be used to promote the regeneration of nerve fibersover long distances following spinal cord damage.

The present invention further relates to neurite growth regulatoryfactor receptors and peptide fragments thereof as well as the nucleicacid sequences coding for neurite growth regulatory factor receptors andfragments, and their therapeutic and diagnostic uses. Antibodies toneurite growth regulatory factor receptors are also envisioned andwithin the scope of the present invention.

The present invention is also directed to metalloproteases associatedwith malignant tumors, in particular, those metastatic to the brain. Ina specific embodiment, the metalloprotease is associated withglioblastoma cells. The metalloproteases of the invention are associatedwith the CNS infiltration activity of malignant cells, and canneutralize the inhibitory substrate properties of the CNSmyelin-associated proteins. The metalloproteases can have therapeuticvalue in the treatment of nerve damage such as that resulting fromtraumatic injury (e.g. spinal cord injuries), stroke, degenerativedisorders of the central nervous system, etc. Alternatively, themetalloprotease may be used in combination with antibodies directedagainst myelin associated inhibitory proteins to treat nerve damage.

The present invention is also directed to inhibitors of themetalloproteases. Such inhibitors can impair malignant cell spreadingand infiltration, and can be used in the treatment of malignant tumors(e.g. glioblastoma). In a specific embodiment, the metalloproteaseinhibitors in combination with CNS myelin associated inhibitory proteinssuch as the 35,000 dalton and/or the 250,000 dalton molecular weightproteins or human functional equivalents thereof, may be used in thediagnosis and/or treatment of malignant tumors which include but are notlimited to glioblastomas, neuroblastomas, and melanomas.

The method of the invention can be divided into the following stages,solely for the purpose of description: (1) isolation and purification ofneurite growth regulatory factors; (2) characterization of neuritegrowth regulatory factors; (3) molecular cloning of genes or genefragment encoding neurite growth regulatory factors; (4) production ofantibodies against neurite growth regulatory factors; and (5) generationof neurite growth regulatory factor related derivatives, analogs, andpeptides. The method further encompasses the diagnostic and therapeuticuses of neurite growth regulatory factors and their antibodies.

5.1. ISOLATION AND PURIFICATION OF NEURITE GROWTH REGULATORY FACTORS

The present invention relates to CNS myelin associated inhibitoryproteins of neurite growth, receptors of CNS myelin associatedinhibitory proteins of neurite growth, and to metalloproteases such asthat associated with membranes of glioblastoma cells. The CNS myelinassociated inhibitory proteins of the invention may be isolated by firstisolating myelin and subsequent purification therefrom. Similarly, themetalloprotease may be obtained by isolation from mammalian glioblastomacells. Isolation procedures which may be employed are described morefully in the sections which follow. Alternatively, the CNS myelinassociated inhibitory proteins or the metalloprotease may be obtainedfrom a recombinant expression system (see Section 5.3., infra).Procedures for the isolation and purification of receptors for the CNSmyelin associated inhibitory proteins are described in Section 5.1.2.,infra.

5.1.1. ISOLATION AND PURIFICATION OF CNS MYELIN ASSOCIATED INHIBITORYPROTEINS

CNS myelin associated inhibitory proteins can be isolated from the CNSmyelin of higher vertebrates including, but not limited to, birds ormammals. Myelin can be obtained from the optic nerve or from centralnervous system tissue that includes but is not limited to spinal cordsor brain stems. The tissue may be homogenized using procedures describedin the art (Colman et al., 1982, J. Cell Biol. 95:598-608). The myelinfraction can be isolated subsequently also using procedures described(Colman et al., 1982, supra).

In one embodiment of the invention, the CNS myelin associated inhibitoryproteins can be solubilized in detergent (e.g., Nonidet P-40™, sodiumdeoxycholate). The solubilized proteins can subsequently be purified byvarious procedures known in the art, including but not limited tochromatography (e.g., ion exchange, affinity, and sizingchromatography), centrifugation, electrophoretic procedures,differential solubility, or by any other standard technique for thepurification of proteins (see, e.g., Section 7.2.3., infra).

Alternatively, the CNS myelin associated inhibitory proteins may beisolated and purified using immunological procedures. For example, inone embodiment of the invention, the proteins can first be solubilizedusing detergent (e.g., Nonidet P-40™, sodium deoxycholate). The proteinsmay then be isolated by immunoprecipitation with antibodies to the 35kilodalton and/or the 250 kilodalton proteins. Alternatively, the CNSmyelin associated inhibitory proteins may be isolated usingimmunoaffinity chromatography in which the proteins are applied to anantibody column in solubilized form.

5.1.2. ISOLATION AND PURIFICATION OF RECEPTORS FOR THE CNS MYELINASSOCIATED INHIBITORY PROTEINS

Receptors for the CNS myelin associated inhibitory proteins can beisolated from cells whose attachment, spreading, growth and/or motilityis inhibited by the CNS myelin associated inhibitory proteins. Suchcells include but are not limited to fibroblasts and neurons. In apreferred embodiment, fibroblasts are used as the source for isolationand purification of the receptors.

In one embodiment, receptors to CNS myelin associated inhibitoryproteins may be isolated by affinity chromatography of fibroblast cellextracts, in which a myelin associated inhibitory protein or peptidefragment thereof is immobilized to a solid support.

5.1.3. ISOLATION AND PURIFICATION OF METALLOPROTEASES ASSOCIATED WITHMALIGNANT TUMORS

The metalloproteases of the present invention may be isolated from cellsof malignant tumors, in particular, those tumors which can metastasizeto the brain. In a preferred embodiment, a metalloprotease can beisolated from mammalian glioblastoma cells. In a preferred method, themetalloprotease is isolated from the plasma membrane fraction of suchcells. The cells may be obtained by dissociating and homogenizing tumorsusing procedures known in the art or from tumor cell lines. Plasmamembrane fractions may be obtained using procedures known in the art,e.g., gradient centrifugation (Quigley, 1976, J. Cell Biol. 71:472-486).The metalloprotease may be isolated from the membranes by solubilizationwith mild ionic or nonionic detergent (e.g., deoxycholate, NonidetP-40™, Triton™, Brij™) using procedures described in the art (reviewedin Cooper, 1977, In Tools of Biochemistry, John Wiley & Sons, NY, pp.355-406).

Purification of the metalloprotease can be carried out by knownprocedures, including but not limited to chromatography (e.g., ionexchange, affinity, and sizing column chromatography), centrifugation,electrophoretic procedures, differential solubility, or by any otherstandard technique for the purification of proteins.

5.2. PROTEIN CHARACTERIZATION

The neurite growth regulatory factors of the present invention can beanalyzed by assays based on their physical, immunological, or functionalproperties. The half life of the neurite growth regulatory factors incultured cells can be studied, for example, by use of cycloheximide, aninhibitor of protein synthesis (Vasquez, 1974, FEBS Lett. 40:563-584).In other experiments, a physiological receptor for a neurite growthregulatory factor could be identified by assays which detect complexformation with a neurite growth regulatory factor, e.g., by use ofaffinity chromatography with immobilized neurite growth regulatoryfactor, binding to a labeled neurite growth regulatory factor followedby cross-linking and immunoprecipitation, etc.

Electrophoretic techniques such as SDS-polyacrylamide gelelectrophoresis and two-dimensional electrophoresis can be used to studyprotein structure. Other techniques which can be used include but arenot limited to peptide mapping, isoelectric focusing, andchromatographic techniques.

The amino acid sequences of primary myelin associated inhibitors or ofthe metalloprotease can be derived by deduction from the DNA sequence ifsuch is available, or alternatively, by direct sequencing of theprotein, e.g., with an automated amino acid sequencer. The proteinsequences can be further characterized by a hydrophilicity analysis(Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828). Ahydrophilicity profile can be used to identify the hydrophobic andhydrophilic regions of the protein (and the corresponding regions of thegene sequence, if available, which encode such regions).

Secondary structural analysis (Chou and Fasman, 1974, Biochemistry13:222) can also be done, to identify regions of the CNS myelinassociated inhibitor or gliobastoma metalloprotease sequence that assumespecific secondary structures. Other methods of structural analysis canalso be employed. These include but are not limited to X-raycrystallography (Engstom, 1974, Biochem. Exp. Biol. 11:7-13) andcomputer modeling (Fletterick, R. and Zoller, M. (eds.), 1986, ComputerGraphics and Molecular Modeling, in Current Communications in MolecularBiology, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

5.3. MOLECULAR CLONING OF GENES OR GENE FRAGMENTS ENCODING NEURITEGROWTH REGULATORY FACTORS 5.3.1. ISOLATION AND CLONING OF THE NEURITEGROWTH REGULATORY FACTOR GENES

Any mammalian cell can potentially serve as the nucleic acid source forthe molecular cloning of the genes encoding the CNS myelin associatedinhibitory proteins, including but not limited to the 35 kD and/or 250kD myelin associated proteins described in Section 7., infra, or theglioblastoma associated metalloprotease, hereinafter referred to asneurite growth regulatory factor genes.

The DNA may be obtained by standard procedures known in the art fromcloned DNA (e.g., a DNA "library"), by chemical synthesis, by cDNAcloning, or by the cloning of genomic DNA, or fragments thereof,purified from the desired mammalian cell. (See, for example, Maniatis etal., 1982, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNACloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K., Vol. I,II.) Clones derived from genomic DNA may contain regulatory and intronDNA regions, in addition to coding regions; clones derived from cDNAwill contain only exon sequences. Whatever the source, a given neuritegrowth regulatory factor gene should be molecularly cloned into asuitable vector for propagation of the gene.

In the molecular cloning of a neurite growth regulatory factor gene fromgenomic DNA, DNA fragments are generated, some of which will encode thedesired neurite growth regulatory factor gene. The DNA may be cleaved atspecific sites using various restriction enzymes. Alternatively, one mayuse DNAse in the presence of manganese to fragment the DNA, or the DNAcan be physically sheared, as for example, by sonication. The linear DNAfragments can then be separated according to size by standardtechniques, including but not limited to, agarose and polyacrylamide gelelectrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing a neurite growth regulatory factor gene may beaccomplished in a number of ways. For example, if an amount of a neuritegrowth regulatory factor gene or its specific RNA, or a fragmentthereof, is available and can be purified and labeled, the generated DNAfragments may be screened by nucleic acid hybridization to the labeledprobe (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness,1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961-3965). For example, in apreferred embodiment, a portion of a neurite growth regulatory factoramino acid sequence can be used to deduce the DNA sequence, which DNAsequence can then be synthesized as an oligonucleotide for use as ahybridization probe. Alternatively, if a purified neurite growthregulatory factor probe is unavailable, nucleic acid fractions enrichedin neurite growth regulatory factor may be used as a probe, as aninitial selection procedure.

It is also possible to identify an appropriate neurite growth regulatoryfactor-encoding fragment by restriction enzyme digestion(s) andcomparison of fragment sizes with those expected according to a knownrestriction map if such is available. Further selection on the basis ofthe properties of the gene, or the physical, chemical, or immunologicalproperties of its expressed product, as described supra, can be employedafter the initial selection.

A neurite growth regulatory factor gene can also be identified by mRNAselection using nucleic acid hybridization followed by in vitrotranslation or translation in Xenopus oocytes. In an example of thelatter procedure, oocytes are injected with total or size fractionatedCNS mRNA populations, and the membrane-associated translation productsare screened in a functional assay (3T3 cell spreading). Preadsorptionof the RNA with complementary DNA (cDNA) pools leading to the absence ofexpressed inhibitory factors indicates the presence of the desired cDNA.Reduction of pool size will finally lead to isolation of a single cDNAclone. In an alternative procedure, DNA fragments can be used to isolatecomplementary mRNAs by hybridization. Such DNA fragments may representavailable, purified neurite growth regulatory factor DNA, or DNA thathas been enriched for neurite growth regulatory factor sequences.Immunoprecipitation analysis or functional assays of the in vitrotranslation products of the isolated mRNAs identifies the mRNA and,therefore, the cDNA fragments that contain neurite growth regulatoryfactor sequences. An example of such a functional assay involves anassay for nonpermissiveness in which the effect of the varioustranslation products on the spreading of 3T3 cells on a polylysinecoated tissue culture dish is observed (see Section 7.1.2., infra). Inaddition, specific mRNAs may be selected by adsorption of polysomesisolated from cells to immobilized antibodies specifically directedagainst a neurite growth regulatory factor protein. A radiolabeledneurite growth regulatory factor cDNA can be synthesized using theselected mRNA (from the adsorbed polysomes) as a template. Theradiolabeled mRNA or cDNA may then be used as a probe to identify theneurite growth regulatory factor DNA fragments from among other genomicDNA fragments.

Alternatives to isolating the neurite growth regulatory factor genomicDNA include, but are not limited to, chemically synthesizing the genesequence itself from a known sequence or making cDNA to the mRNA whichencodes the neurite growth regulatory factor gene. Other methods arepossible and within the scope of the invention.

The identified and isolated gene or cDNA can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, cosmids, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pBR322 or pUC plasmid derivatives. Recombinant molecules can beintroduced into host cells via transformation, transfection, infection,electroporation, etc.

In an alternative embodiment, the neurite growth regulatory factor genemay be identified and isolated after insertion into a suitable cloningvector, in a "shot gun" approach. Enrichment for a given neurite growthregulatory factor gene, for example, by size fractionation orsubtraction of cDNA specific to low neurite growth regulatory factorproducers, can be done before insertion into the cloning vector. Inanother embodiment, DNA may be inserted into an expression vectorsystem, and the recombinant expression vector containing a neuritegrowth regulatory factor gene may then be detected by functional assaysfor the neurite growth regulatory factor protein.

The neurite growth regulatory factor gene is inserted into a cloningvector which can be used to transform, transfect, or infect appropriatehost cells so that many copies of the gene sequences are generated. Thiscan be accomplished by ligating the DNA fragment into a cloning vectorwhich has complementary-cohesive termini. However, if the complementaryrestriction sites used to fragment the DNA are not present in thecloning vector, the ends of the DNA molecules may be enzymaticallymodified. Alternatively, any site desired may be produced by ligatingnucleotide sequences (linkers) onto the DNA termini; these ligatedlinkers may comprise specific chemically synthesized oligonucleotidesencoding restriction endonuclease recognition sequences. In analternative method, the cleaved vector and neurite growth regulatoryfactor gene may be modified by homopolymeric tailing.

Identification of the cloned neurite growth regulatory factor gene canbe accomplished in a number of ways based on the properties of the DNAitself, or alternatively, on the physical, immunological, or functionalproperties of its encoded protein. For example, the DNA itself may bedetected by plaque or colony nucleic acid hybridization to labeledprobes (Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M.and Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961).Alternatively, the presence of a neurite growth regulatory factor genemay be detected by assays based on properties of its expressed product.For example, cDNA clones, or DNA clones which hybrid-select the propermRNAs, can be selected which produce a protein that inhibits in vitroneurite outgrowth. If an antibody to a neurite growth regulatory factoris available, a neurite growth regulatory factor protein may beidentified by binding of labeled antibody to the putatively neuritegrowth regulatory factor-synthesizing clones, in an ELISA (enzyme-linkedimmunosorbent assay)-type procedure.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate an isolated neurite growth regulatoryfactor gene, cDNA, or synthesized DNA sequence enables generation ofmultiple copies of the gene. Thus, the gene may be obtained in largequantities by growing transformants, isolating the recombinant DNAmolecules from the transformants and, when necessary, retrieving theinserted gene from the isolated recombinant DNA.

If the ultimate goal is to insert the gene into virus expression vectorssuch as vaccinia virus or adenovirus, the recombinant DNA molecule thatincorporates a neurite growth regulatory factor gene can be modified sothat the gene is flanked by virus sequences that allow for geneticrecombination in cells infected with the virus so that the gene can beinserted into the viral genome.

After the neurite growth regulatory factor DNA-containing clone has beenidentified, grown, and harvested, its DNA insert may be characterized asdescribed in Section 5.3.4, infra. When the genetic structure of aneurite growth regulatory factor gene is known, it is possible tomanipulate the structure for optimal use in the present invention. Forexample, promoter DNA may be ligated 5' of a neurite growth regulatoryfactor coding sequence, in addition to or replacement of the nativepromoter to provide for increased expression of the protein. Manymanipulations are possible, and within the scope of the presentinvention.

5.3.2. EXPRESSION OF THE CLONED NEURITE GROWTH REGULATORY FACTOR GENES

The nucleotide sequence coding for a neurite growth regulatory factorprotein or a portion thereof, can be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted protein-codingsequence. The necessary transcriptional and translation signals can alsobe supplied by the native neurite growth regulatory factor gene and/orits flanking regions. A variety of host-vector systems may be utilizedto express the protein-coding sequence. These include but are notlimited to mammalian cell systems infected with virus (e.g., vacciniavirus, adenovirus, etc.); insect cell systems infected with virus (e.g.,baculovirus); microorganisms such as yeast containing yeast vectors, orbacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.The expression elements of these vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a chimeric gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinations (genetic recombination).

Expression vectors containing neurite growth regulatory factor geneinserts can be identified by three general approaches: (a) DNA--DNAhybridization, (b) presence or absence of "marker" gene functions, and(c) expression of inserted sequences. In the first approach, thepresence of a foreign gene inserted in an expression vector can bedetected by DNA--DNA hybridization using probes comprising sequencesthat are homologous to an inserted neurite growth regulatory factorgene. In the second approach, the recombinant vector/host system can beidentified and selected based upon the presence or absence of certain"marker" gene functions (e.g., thymidine kinase activity, resistance toantibiotics, transformation phenotype, occlusion body formation inbaculovirus, etc.) caused by the insertion of foreign genes in thevector. For example, if a given neurite growth regulatory factor gene isinserted within the marker gene sequence of the vector, recombinantscontaining the neurite growth regulatory factor insert can be identifiedby the absence of the marker gene function. In the third approach,recombinant expression vectors can be identified by assaying the foreigngene product expressed by the recombinant. Such assays can be based onthe physical, immunological, or functional properties of a given neuritegrowth regulatory factor gene product.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors which can be used include,but are not limited to, the following vectors or their derivatives:human or animal viruses such as vaccinia virus or adenovirus; insectviruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g.,lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered neurite growth regulatoryfactor protein may be controlled. Furthermore, different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification (e.g., glycosylation,cleavage) of proteins. Appropriate cell lines or host systems can bechosen to ensure the desired modification and processing of the foreignprotein expressed. For example, expression in a bacterial system can beused to produce an unglycosylated core protein product. Expression inyeast will produce a glycosylated product. Expression in mammalian (e.g.COS) cells can be used to ensure "native" glycosylation of theheterologous neurite growth regulatory factor protein. Furthermore,different vector/host expression systems may effect processing reactionssuch as proteolytic cleavages to different extents.

5.3.3. IDENTIFICATION AND PURIFICATION OF THE EXPRESSED GENE PRODUCT

Once a recombinant which expresses a given neurite growth regulatoryfactor gene is identified, the gene product can be purified as describedin Section 5.1, supra, and analyzed as described in Section 5.2, supra.

The amino acid sequence of a given neurite growth regulatory factorprotein can be deduced from the nucleotide sequence of the cloned gene,allowing the protein, or a fragment thereof, to be synthesized bystandard chemical methods known in the art (e.g., see Hunkapiller, etal., 1984, Nature 310:105-111).

In particular embodiments of the present invention, such neurite growthregulatory factor proteins, whether produced by recombinant DNAtechniques or by chemical synthetic methods, include but are not limitedto those containing altered sequences in which functionally equivalentamino acid residues are substituted for residues within the sequenceresulting in a silent change. For example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofa similar polarity which acts as a functional equivalent, resulting in asilent alteration. Substitutes for an amino acid within the sequence maybe selected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine. The polar neutral amino acids includeglycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine, and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Also included within the scopeof the invention are neurite growth regulatory factor proteins which aredifferentially modified during or after translation, e.g., byglycosylation, proteolytic cleavage, etc.

5.3.4. CHARACTERIZATION OF THE NEURITE, GROWTH REGULATORY FACTOR GENES

The structure of a given neurite growth regulatory factor gene can beanalyzed by various methods known in the art.

The cloned DNA or cDNA corresponding to a given neurite growthregulatory factor gene can be analyzed by methods including but notlimited to Southern hybridization (Southern, 1975, J. Mol. Biol.98:503-517), Northern hybridization (Alwine, et al., 1977, Proc. Natl.Acad. Sci. U.S.A. 74:5350-5354; Wahl, et al., 1987, Meth. Enzymol.152:572-581), restriction endonuclease mapping (Maniatis, et al., 1982,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.), and DNA sequence analysis.

DNA sequence analysis can be performed by any techniques known in theart including but not limited to the method of Maxam and Gilbert (1980,Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger, et al.,1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467), or use of anautomated DNA sequenator (e.g., Applied Biosystems, Foster City,Calif.).

5.4. PRODUCTION OF ANTIBODIES TO NEURITE, GROWTH REGULATORY FACTORS

Antibodies can be produced which recognize neurite growth regulatoryfactors or related proteins. Such antibodies can be polyclonal ormonoclonal.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to epitopes of a given neurite growth regulatoryfactor. For the production of antibody, various host animals can beimmunized by injection with a neurite growth regulatory factor protein,or a synthetic protein, or fragment thereof, including but not limitedto rabbits, mice, rats, etc. Various adjuvants may be used to increasethe immunological response, depending on the host species, and includingbut not limited to Freund's (complete and incomplete), mineral gels suchas aluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and corynebacterium parvum.

A monoclonal antibody to an epitope of a neurite growth regulatoryfactor can be prepared by using any technique which provides for theproduction of antibody molecules by continuous cell lines in culture.These include but are not limited to the hybridoma technique originallydescribed by Kohler and Milstein (1975, Nature 256:495-497), and themore recent human B cell hybridoma technique (Kozbor et al., 1983,Immunology Today 4:72) and EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). In a particular embodiment, the procedure described infra inSection 8.1. may be used to obtain mouse monoclonal antibodies whichrecognize the 35 kD and 250 kD CNS myelin associated inhibitoryproteins.

The monoclonal antibodies for therapeutic use may be human monoclonalantibodies or chimeric human-mouse (or other species) monoclonalantibodies. Human monoclonal antibodies may be made by any of numeroustechniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad.Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79;Olsson et al., 1982, Meth. Enzymol. 92:3-16). Chimeric antibodymolecules may be prepared containing a mouse antigen-binding domain withhuman constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci.U.S.A. 81:6851, Takeda et al., 1985, Nature 314:452).

A molecular clone of an antibody to a neurite growth regulatory factorepitope can be prepared by known techniques. Recombinant DNA methodology(see e.g., Maniatis et al., 1982, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) may beused to construct nucleic acid sequences which encode a monoclonalantibody molecule, or antigen binding region thereof.

Antibody molecules may be purified by known techniques, e.g.,immunoabsorption or immunoaffinity chromatography, chromatographicmethods such as HPLC (high performance liquid chromatography), or acombination thereof, etc.

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab')₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab' fragments which can begenerated by reducing the disulfide bridges of the F(ab')₂ fragment, andthe 2 Fab or Fab fragments which can be generated by treating theantibody molecule with papain and a reducing agent.

5.5. NEURITE GROWTH REGULATORY FACTOR-RELATED DERIVATIVES, ANALOGS, ANDPEPTIDES

The production and use of derivatives, analogs, and peptides related toa given neurite growth regulatory factor are also envisioned, and withinthe scope of the present invention and include molecules antagonistic toneurite growth regulatory factors (for example, and not by way oflimitation, anti-idiotype antibodies). Such derivatives, analogs, orpeptides which have the desired inhibitory activity can be used, forexample, in the treatment of neuroblastoma (see Section 5.6, infra).Derivatives, analogs, or peptides related to a neurite growth regulatoryfactor can be tested for the desired activity by assays fornonpermissive substrate effects. For example, procedures such as theassay for nonpermissiveness in which the effect of the varioustranslation products on the spreading of 3T3 cells on a polylysinecoated tissue culture dish is observed (see Section 7.1.2., infra).

The neurite growth regulatory factor-related derivatives, analogs, andpeptides of the invention can be produced by various methods known inthe art. The manipulations which result in their production can occur atthe gene or protein level. For example, a cloned neurite growthregulatory factor gene can be modified by any of numerous strategiesknown in the art (Maniatis, et al., 1982, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.). A given neurite growth regulatory factor sequence can be cleavedat appropriate sites with restriction endonuclease(s), subjected toenzymatic modifications if desired, isolated, and ligated in vitro. Inthe production of a gene encoding a derivative, analogue, or peptiderelated to a neurite growth regulatory factor, care should be taken toensure that the modified gene remains within the same translationalreading frame as the neurite growth regulatory factor, uninterrupted bytranslational stop signals, in the gene region where the desired neuritegrowth regulatory factor-specific activity is encoded.

Additionally, a given neurite growth regulatory factor gene can bemutated in vitro or in vivo, to create and/or destroy translation,initiation, and/or termination sequences, or to create variations incoding regions and/or form new restriction endonuclease sites or destroypreexisting ones, to facilitate further in vitro modification. Anytechnique for mutagenesis known in the art can be used, including butnot limited to, in vitro site-directed mutagenesis (Hutchinson, et al.,1978, J. Biol. Chem. 253:6551), use of TABS linkers (Pharmacia), etc.

5.6. USES OF NEURITE GROWTH REGULATORY FACTORS 5.6.1. DIAGNOSTIC USES5.6.1.1. CNS MYELIN ASSOCIATED INHIBITORY PROTEINS

CNS myelin associated inhibitory proteins, analogs, derivatives, andsubsequences thereof, and anti-inhibitory protein antibodies have usesin diagnostics. Such molecules can be used in assays such asimmunoassays to detect, prognose, diagnose, or monitor variousconditions, diseases, and disorders affecting neurite growth extension,invasiveness, and regeneration. In one embodiment of the invention,these molecules may be used for the diagnosis of malignancies.Alternatively, the CNS myelin associated inhibitory proteins, analogs,derivatives, and subsequences thereof may be used to monitor therapiesfor diseases and conditions which ultimately result in nerve damage;such diseases and conditions include but are not limited to CNS trauma,(e.g. spinal cord injuries), infarction, infection, malignancy, exposureto toxic agents, nutritional deficiency, paraneoplastic syndromes, anddegenerative nerve diseases (including but not limited to Alzheimer'sdisease, Parkinson's disease, Huntington's Chorea, amyotrophic lateralsclerosis, and progressive supra-nuclear palsy). In a specificembodiment, such molecules may be used to detect an increase in neuriteoutgrowth as an indicator of CNS fiber regeneration.

For example, in specific embodiments, the absence of the CNS myelinassociated inhibitory proteins in a patient sample containing CNS myelincan be a diagnostic marker for the presence of a malignancy, includingbut not limited to glioblastoma, neuroblastoma, and melanoma, or acondition involving nerve growth, invasiveness, or regeneration in apatient. In a particular embodiment, the absence of the inhibitoryproteins can be detected by means of an immunoassay in which the lack ofany binding to anti-inhibitory protein antibodies (e.g., IN-1, IN-2) isobserved.

The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"immunoassays, precipitation reactions, gel diffusion precipitationreactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, immunoelectrophoresis assays, andimmunohistochemistry on tissue sections, to name but a few.

In a specific embodiment, ligands which bind to a CNS myelin associatedinhibitory protein can be used in imaging techniques. For example, smallpeptides (e.g., inhibitory protein receptor fragments) which bind to theinhibitory proteins, and which are able to penetrate through theblood-brain barrier, when labeled appropriately, can be used for imagingtechniques such as PET (positron emission tomography) diagnosis orscintigraphy detection, under conditions noninvasive to the patient.

Neurite growth inhibitory factor genes, DNA, cDNA, and RNA, and relatednucleic acid sequences and subsequences, including complementarysequences, can also be used in hybridization assays. The neurite growthinhibitory factor nucleic acid sequences, or subsequences thereofcomprising about at least 15 nucleotides, can be used as hybridizationprobes. Hybridization assays can be used to detect, prognose, diagnose,or monitor conditions, disorders, or disease states associated withchanges in neurite growth inhibitory factor expression as describedsupra. For example, total RNA in myelin, e.g., on biopsy tissuesections, from a patient can be assayed for the presence of neuritegrowth inhibitory factor mRNA, where the amount of neurite growthinhibitory factor mRNA is indicative of the level of inhibition ofneurite outgrowth activity in a given patient.

5.6.1.2. CNS MYELIN ASSOCIATED INHIBITORY PROTEIN RECEPTORS

CNS myelin associated inhibitory protein receptors as well as analogs,derivatives, and subsequences thereof, and anti-receptor antibodies haveuses in diagnostics. These molecules of the invention can be used inassays such as immunoassays or binding assays to detect, prognose,diagnose, or monitor various conditions, diseases, and disordersaffecting neurite growth, extension, invasion, and regeneration. Forexample, it is possible that a lower level of expression of thesereceptors may be detected in various disorders associated with enhancedneurite invasiveness or regeneration such as those involving nervedamage, infarction, degenerative nerve diseases, or malignancies. TheCNS myelin associated inhibitory protein receptors, analogs,derivatives, and subsequences thereof may also be used to monitortherapies for diseases and disorders which ultimately result in nervedamage, which include but are not limited to CNS trauma (e.g. spinalcord injuries), stroke, degenerative nerve diseases, and formalignancies.

The assays which can be used include but are not limited to thosedescribed supra in Section 5.6.1.1.

Neurite growth inhibitory factor receptor genes and related nucleic acidsequences and subsequences, including complementary sequences, can alsobe used in hybridization assays, to detect, prognose, diagnose, ormonitor conditions, disorders, or disease states associated with changesin neurite growth inhibitory factor receptor expression.

5.6.1.3. METALLOPROTEASES AND THEIR INHIBITORS

The metalloproteases of the invention, and their analogs, derivatives,and fragments thereof, as well as inhibitors and anti-metalloproteaseantibodies, may be used for diagnostic purposes. These molecules of theinvention may be used in assays such as immunoassays or inhibition typeassays to detect, prognose, diagnose, or monitor various conditions,diseases, and disorders affecting neurite growth extension,invasiveness, or regeneration. In a specific embodiment, the moleculesof the present invention can be used to diagnose malignant tumors, inparticular, those capable of metastasizing to the brain, (e.g.,glioblastoma) by detecting the presence of or an increase inmetalloprotease levels. Alternatively, the molecules of the presentinvention may be used to monitor therapies for malignant tumors such asglioblastoma by detecting changes in metalloprotease levels. In thislatter embodiment, decreases or the disappearance of metalloproteaselevels should can be indicative of treatment efficacy.

The assays which can be used include but are not limited to thosedescribed supra in Section 5.6.1.1.

Metalloprotease genes and related nucleic acid sequences andsubsequences, including complementary sequences, can also be used inhybridization assays, to detect, prognose, diagnose, or monitorconditions, disorders, or disease states associated with changes inmetalloprotease expression as described supra. For example, total RNA ina sample (e.g., glial cells) from a patient can be assayed for thepresence of metalloprotease mRNA, where the presence or amount ofmetalloprotease mRNA is indicative of a malignancy in the patient. Inparticular, a malignancy that can be metastatic to the brain (e.g.,glioblastoma) can be detected.

5.6.2. THERAPEUTIC USES 5.6.2.1. CNS MYELIN ASSOCIATED INHIBITORYPROTEINS

CNS myelin associated inhibitory proteins of the present invention canbe therapeutically useful in the treatment of patients with malignanttumors including, but not limited to melanoma or tumors of nerve tissue(e.g. neuroblastoma). In one embodiment, patients with neuroblastoma canbe treated with the 35 kD and/or 250 kD proteins or analogs,derivatives, or subsequences thereof, and the human functionalequivalents thereof, which are inhibitors of neurite extension. Inanother embodiment, a patient can be therapeutically administered both aCNS myelin-associated inhibitory protein and a metalloproteaseinhibitor.

In an alternative embodiment, derivatives, analogs, or subsequences ofCNS myelin inhibitory proteins which inhibit the native inhibitoryprotein function can be used in regimens where an increase in neuriteextension, growth, or regeneration is desired, e.g., in patients withnerve damage. Patients suffering from traumatic disorders (including butnot limited to spinal cord injuries, spinal cord lesions, or other CNSpathway lesions), surgical nerve lesions, damage secondary toinfarction, infection, exposure to toxic agents, malignancy,paraneoplastic syndromes, or patients with various types of degenerativedisorders of the central nervous system (Cutler, 1987, In: ScientificAmerican Medicines v. 2, Scientific American Inc., NY, pp. 11-1-11-13)can be treated with such inhibitory protein antagonists. Examples ofsuch disorders include but are not limited to Alzheimer's Disease,Parkinsons' Disease, Huntington's Chorea, amyotrophic lateral sclerosis,or progressive supranuclear palsy. Such antagonists may be used topromote the regeneration of CNS pathways, fiber systems and tracts.Administration of antibodies directed to an epitope of CNS myelinassociated inhibitory proteins such as the 35 kD and/or 250 kD proteins,(or the binding portion thereof, or cells secreting such as antibodies)can also be used to inhibit 35 kD and/or 250 kD protein function inpatients. In a particular embodiment of the invention, antibodiesdirected to the 35 kD and/or 250 kD myelin associated inhibitory proteinmay be used to promote the regeneration of nerve fibers over longdistances following spinal cord damage; in a specific example,monoclonal antibody IN-1 may be used.

Various delivery systems are known and can be used for delivery of CNSmyelin inhibitory proteins, related molecules, or antibodies thereto,e.g., encapsulation in liposomes or semipermeable membranes, expressionby bacteria, etc. Linkage to ligands such as antibodies can be used totarget myelin associated protein-related molecules to therapeuticallydesirable sites in vivo. Methods of introduction include but are notlimited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, oral, and intranasal routes, and infusion into ventriclesor a site of tumor removal. Likewise, cells secreting CNS myelininhibitory protein antagonist activity, for example, and not by way oflimitation, hybridoma cells, encapsulated in a suitable biologicalmembrane may be implanted in a patient so as to provide a continuoussource of anti-CNS myelin inhibiting protein antibodies.

In addition, any method which results in decreased synthesis of CNSmyelin inhibitory proteins may be used to diminish their biologicalfunction. For example, and not by way of limitation, agents toxic to thecells which synthesize CNS myelin inhibitory proteins (e.g.oligodendrocytes) may be used to decrease the concentration ofinhibitory proteins to promote regeneration of neurons.

5.6.2.2. CNS MYELIN ASSOCIATED INHIBITORY PROTEIN RECEPTORS

CNS myelin associated inhibitory protein receptors or fragments thereof,and antibodies thereto, can be therapeutically useful in the treatmentof patients with nerve damage including but not limited to thatresulting from CNS trauma (e.g., spinal cord injuries), infarction, ordegenerative disorders of the central nervous system which include butare not limited to Alzheimer's disease, Parkinson's disease,Huntington's Chorea, amyotrophic lateral sclerosis, or progressivesupranuclear palsy. For example, in one embodiment, CNS myelinassociated inhibitory protein receptors, or subsequences or analogsthereof which contain the inhibitory protein binding site, can beadministered to a patient to "compete out" binding of the inhibitoryproteins to their natural receptor, and to thus promote nerve growth orregeneration in the patient. In an alternative embodiment, antibodies tothe inhibitory protein receptor (or the binding portion thereof or cellssecreting antibodies binding to the receptor) can be administered to apatient in order to prevent receptor function and thus promote nervegrowth or regeneration in the patient. Patients in whom such a therapymay be desired include but are not limited to those with nerve damage,stroke, or degenerative disorders of the central nervous system asdescribed supra.

Various delivery systems are known and can be used for delivery of CNSmyelin associated inhibitory protein receptors, related molecules, orantibodies thereto, e.g., encapsulation in liposomes, expression bybacteria, etc. Linkage to ligands such as antibodies can be used totarget myelin associated protein receptor-related molecules totherapeutically desirable sites in vivo. Methods of introduction includebut are not limited to intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, oral, intranasal routes, and infusion intoventricles or a site of tumor removal.

5.6.2.3. METALLOPROTEASES AND THEIR INHIBITORS

The metalloproteases of the present invention can be therapeuticallyuseful in the treatment of various types of nerve damage or degenerativedisorders of the central nervous system (such as those described supra,Section 5.6.2.2) In one embodiment, patients suffering from nerve damageresulting from trauma, stroke, or neurodegenerative disorders can betreated with the metalloprotease or proteolytically active analogs,derivatives, or subsequences thereof which stimulate neurite extensionor regeneration of CNS fiber.

In an alternative embodiment, derivatives, analogs, or subsequences ofthe metalloproteases which antagonize or inhibit metalloproteasefunction, or chemical inhibitors of the metalloprotease activity, can beused in regimens where an inhibition of invasive migration and spread inthe CNS is desired. Such inhibitors may include but are not limited to1,10 phenanthroline, EDTA, EGTA, cbz-tyr-tyr, cbz-gly-phe-NH₂,cbz-phe-phe-NH₂, and cbz-gly-phe-phe-NH₂. 1,10 phenanthroline, EDTA, andEGTA may be obtained from commercial vendors (e.g. Sigma Chemical Co.).Cbz-tyr-tyr, cbz-gly-phe-NH₂, cbz-phe-phe-NH₂, and cbz-gly-phe-phe-NH₂may also be obtained from commercial vendors (e.g. VegaBiotechnologies), or may be chemically synthesized. In specificembodiments, patients with various types of malignant tumors, inparticular, those metastatic to the brain, can be treated with suchinhibitors. In a preferred embodiment, a patient with a glioblastoma canbe treated with such inhibitors. In another specific embodiment,administration of antibodies directed to an epitope of themetalloprotease can also be used to inhibit metalloprotease function inpatients. In yet another specific embodiment of the invention,metalloprotease inhibitors and a CNS myelin associated inhibitoryprotein can both be administered to a patient for the treatment of amalignant tumor, examples of which include but are not limited toglioblastoma, neuroblastoma, or a melanoma.

Various delivery systems are known and can be used for the delivery ofmetalloproteases and related molecules, e.g., encapsulation in liposomesor semipermeable membranes, expression by bacteria, etc. Linkage toligands such as antibodies can be used to target molecules totherapeutically desirable sites in vivo. Methods of introduction includebut are not limited to intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, oral, and intranasal routes, and infusioninto ventricles or a site of tumor removal.

6. OLIGODENDROCYTES AND CNS MYELIN ARE NONPERMISSIVE SUBSTRATES FORNEURITE GROWTH AND FIBROBLAST SPREADING IN VITRO

To study the interaction of neurons with central nervous system (CNS)glial cells, dissociated sympathetic or sensory ganglion cells or fetalretinal cells were plated onto cultures of dissociated optic nerve glialcells of young rats. Whereas astrocytes favored neuron adhesion andneurite outgrowth, oligodendrocytes differed markedly in theirproperties as neuronal substrates. Immature (O₄ ⁺, A₂ B₅ ⁺, GalC⁻),oligodendrocytes were frequently contacted by neurons and neurites. Incontrast, differentiated oligodendrocytes (O₄ ⁺, A₂ B₅ ⁻, GalC⁺)represented a nonpermissive substrate for neuronal adhesion and neuritegrowth. When neuroblastoma cells or 3T3 fibroblasts were plated intooptic nerve glial cultures, the same differences were observed;differentiated oligodendrocytes were nonpermissive for cell adhesion,neurite growth, or fibroblast spreading. These nonpermissiveoligodendrocytes were characterized by a radial, highly branched processnetwork, often contained myelin basic protein (MBP), and may, therefore,correspond to cells actively involved in the production of myelin-likemembranes.

Isolated myelin from adult rat spinal cord was adsorbed to polylysinecoated culture dishes and tested as substrate for peripheral neurons,neuroblastoma cells, or 3T3 cells. Again, cell attachment, neuriteoutgrowth, and fibroblast spreading was strongly impaired. Generalphysico-chemical properties of myelin were not responsible for thiseffect, since myelin from rat sciatic nerves favored neuron adhesion andneurite growth as well as spreading of 3T3 cells. These results showthat differentiated oligodendrocytes express non-permissive substrateproperties, which may be of importance in CNS development orregeneration.

6.1. MATERIALS AND METHODS 6.1.1. GLIAL CELL CULTURES

Optic nerves were dissected from 7-12 day old or young adult (180-220 g)Wistar rats and collected in plating medium (air-buffered enriched L₁₅with 5% rat serum; Mains and Patterson, 1973, J. Cell Biol. 59:329-345).The meninges and blood vessels were carefully removed under a microscopeand the nerves were cut into small pieces. Dissociation of 10 day oldnerves was done for 25 minutes twice in 0.25% trypsin (Sigma) and 0.02%collagenase (Worthington) (Raff et al., 1979, Brain Res. 174:283-318) inCMF-PBS (Ca⁺⁺ /Mg⁺⁺ -free phosphate buffered saline) at 370C. Adultoptic nerves were dissociated in 0.1% trypsin, 0.1% collagenase for 1hour at 37° C. followed by 0.5% trypsin for 10 minutes. After washingand dissociation by trituration with a Pasteur pipet, the cells wereplated into the wells of 35 mm tissue culture dishes containing fourinternal wells at a density of 20,000 to 30,000 cells per well (surfaceof well: 95 mm²). For 7-10 day old optic nerves the yield of thedissociation was about 10,000 cells per nerve. The culture substrate formost of the experiments was polyornithine (PORN, Sigma, 0.5 mg/ml inborate buffer, incubated overnight) or polylysine (PLYS, Sigma, 50 ng/mlin water); in some experiments, a dried collagen film (calf skincollagen, incubation overnight with sterile solution), laminin-coatedPORN (purified mouse EHS tumor laminin (5 ng/ml, incubated for 3 hourson dishes previously coated with PORN), or plain tissue culture plasticwas used. The culture medium was an enriched L₁₅ medium with 5% ratserum, penicillin (100 U/ml) and streptomycin (100 ng/ml) (Mains andPatterson, 1973, J. Cell Biol. 59:329-345). In some experiments, 10%fetal calf serum (FCS) was added instead of the rat serum.

Optic nerves of E13 or E17 chicken embryos were dissociated by brieftrypsin/collagenase treatment and cultured for 2-7 days in L₁₅ with 5%FCS on PORN-coated culture dishes.

6.1.2. GLIA--NERVE CELL CO-CULTURES

Three different types of nerve cells were co-cultured with glial cells:sympathetic neurons from the superior cervical ganglion of newborn rats,sensory neurons from dorsal root ganglia of newborn rats, or cells fromthe retina of E17-E18 embryonic rats. Superior cervical and dorsal rootganglia were dissected and dissociated into single cells as described(Mains and Patterson, 1973, J. Cell Biol. 59:329-345; Schwab andThoenen, 1985, J. Neurosci. 5:2415-2423). Retinas were dissected fromthe embryos, cleaned from adhering blood vessels and incubated in 0.03%trypsin, 0.03% DNase for 10 minutes at 37° C., washed by centrifugationin serum-containing medium and dissociated by trituration.

The neurons were added to 2-10 day old glial cultures in the samemedium, with the addition of NGF (2.5S NGF, 50 or 100 ng/ml) for sensoryand sympathetic neurons or brain-derived neurotrophic factor for theretinal cells (Johnson, J. E. et al., 1986, J. Neurosci. 6:3031-3038).In order to suppress the growth of Schwann cells added together with theperipheral neurons, pulses of cytosine arabinoside (Ara C, 10⁻⁵ M) weregiven twice for 24 hours on the 2nd and 5th day of co-culture in someexperiments. The cultures were processed for antibody staining after 1-5days of co-culture in the case of retina cells, or after 2 days to 3weeks in the case of peripheral ganglion cells.

Mouse neuroblastoma cells (line NB-2A) cultured in DMEM/10% FCS weredetached from the culture flasks by a brief treatment with 0.1% trypsinin CMF-Hank's solution terminated by addition of DMEM/FCS. Afterwashing, the cells were added to glial cultures (40,000 or 20,000 cellsper well) in DMEM/FCS with either 2 mM dibutyryl-cyclic AMP orglia-derived neurite promoting factor (GdNPF; Guenther et al., 1986,EMBO J. 4:1963-1966).

Mouse NIH 3T3 cells, treated identically to the neuroblastoma cells,were added to 2-3 day old cultures of 7 day old or newborn rat opticnerves at a concentration of 20,000 or 40,000 cells per well in DMEMcontaining 10% fetal calf serum or in MEM supplied with insulin (20ng/ml) and transferrin (50 ng/ml). Cultures were returned to theincubator for 2-4 hours and then fixed with warm 4% formalin inphosphate buffer and double stained with the O₁ and O₄ antibodies.

6.1.3. IMMUNOFLUORESCENCE

The following antibodies as markers for oligodendrocytes, astrocytes,neurons or fibroblasts were used: oligodendrocytes: mouse monoclonalantibody (mAB) O₄ (Sommer and Schachner, 1981, Dev. Biol. 83:311-327);mouse mAB O₁ (Sommer and Schachner, 1981, Dev. Biol. 83:311-327);specific for galactocerebroside (GalC; Singh and Pfeiffer, 1985, J.Neurochem. 45:1371-1381); goat antiserum against myelin basic protein ofrabbits (Omlin, et al., 1982, J. Cell Biol. 95:242-248). Precursorcells: mouse mAB A₂ B₅ (Sera-Lab, Crawley Down, GB). Astrocytes: rabbitantiserum against glial fibrillary acid protein (GFAP) (Dahl andBignami, 1976, Brain Res. 116:150-157). Neurons: mouse mAB againstguinea pig or rabbit neurofilaments (Willard and Simon, 1981, J. CellBiol. 89:198-205). Fibroblasts: mouse mAB O×7 against Thy-1.1(Sera-Lab); goat antiserum against human fibronectin (LETS protein;Cappel, N.C.).

The specific antibodies were visualized by the corresponding anti-mouse,anti-rabbit or anti-goat--fluorescein isothiocyanate (FITC)or--rhodamine isothiocyanate (RITC) linked secondary antibodies (Cappel,N.C.). Prior to staining, the cultures were washed twice with PBScontaining 5% sucrose and 0.1% bovine serum albumin (BSA). Theantibodies O₁, O₄ and A₂ B₅ were directed against surface antigens andwere therefore incubated on the living cultures at room temperature for30 minutes at a dilution of 1:20 in PBS/sucrose/BSA. Antibodies againstThy-1 were diluted 1:10, anti-fibronectin 1:20. The cultures were thenrinsed twice, fixed for 10 minutes with 4% formalin in PBS, rinsedagain, incubated for 1 hour with the labeled secondary antibodies(dilution 1:30 to 1:100), washed and mounted in PBS:glycerol (1:1).--Forvisualization of myelin basic protein (MBP) the cultures were brieflyfixed in 4% formalin, then treated with ethanol/acetic acid and finallyincubated with anti-MBP antiserum (1:500 dilution) for 1 hour at roomtemperature. Ethanol/acetic acid fixation was also used forvisualization of neurofilaments. For double labeling experiments of A₂B₅ or O₁ antibodies with the O₄ antibody, living cultures were firstincubated with antibodies A₂ B₅ or O₁ followed by anti-mouse-FITC, andthen with antibody O₄ antigen; the sequence was reversed in someexperiments. Staining the GFAP was done on cultures previously fixed in95% ethanol/5% acetic acid for 30 minutes at 4° C. and rehydrated intoPBS. In the case of O₄ /GFAP double-labeling experiments, staining wasfirst done with the O₄ antibody on the living cultures followed by 10minutes fixation in 4% formalin, subsequent ethanol/acetic acidtreatment and GFAP-staining. For visualization of MBP, the clutures werebriefly fixed in 4% formalin, then treated with ethanol/acetic acid andfinally incubated with anti-MBP antiserum (1:500) for one hour at roomtemperature. Ethanol/acetic acid fixation was also used forvisualization of neurofilaments.

Double-labeled cultures were evaluated by systematically screening inthe fluorescence microscope for the presence of one antigen (usuallyO₄), and every labeled cell was examined for the presence of the otherantigen, e.g. A₂ B₅, O₁, or GFAP.

6.1.4. EVALUATION OF CO-CULTURES WITH NERVE Cells, Neuroblastoma Cells,or 3T3 Cells

Antibody-labeled cultures were systematically screened in thefluorescence microscope and all O₄ -labeled cells were photographed. Thesame fields were photographed under phase contrast illumination. Theoligodendrocyte surface area occupied by or in contact with neurons,neurites, ganglionic Schwann cells, or 3T3 cells was estimated and theoligodendrocytes were grouped into 3 categories: cells with <20%,20%-80%, or >80% of the territory covered by neurons, neurites or 3T3cells. Single thin processes, especially of immature cells, were oftenexcluded from the evaluation for reason of comparability with the denseprocess network of highly branched oligodendrocytes. In experiments withretinal cells, total oligodendrocyte territory and areas overlapped byretinal cells were measured with a Hewlett-Packard digitizer. Theoligodendrocyte subtypes were identified on the correspondingfluorescence micrographs. The criteria used for identification were cellmorphology and antigenic characteristics (O₄ /O₁). A₂ B₅ -staining couldnot be used as a marker for immature cells, since this antigen wasrapidly lost (without a concomitant change in cell morphology); aftercoculture with neurons. The distinguishing morphological criteria were:shape and size of the cell body, number of primary processes, branchingpattern of processed, and the occurrence of anastomoses and membranesheets within the process network. With these criteria, highly branchedoligodendrocytes and immature oligodendrocytes could be reproduciblydistinguished. Most (but not all) of the highly branched cells werepositive for the O₁ antigen; immature cells were consistently negative.

Quantification of the direction of neuroblastoma process outgrowth withrespect to highly branched oligodendrocytes was done as illustrated inFIG. 5. Highly branched oligodendrocytes were sampled systematically,and neighbouring neuroblastoma cells were classified as "adjacent" ifthe distance between the edge of the oligodendrocyte process network andthe NB-2A cell was less than 2 cell body diameters. Further cells wereclassified as "distant" (FIGS. 4A-F and 5). A circle with 8 sectors (4classes) was overlaid over the center of each neuroblastoma cell,oriented towards the nearest oligodendrocyte cell body, and theneuroblastoma processes counted in each sector (FIG. 5).

6.1.5. PREPARATION OF MYELIN

Spinal cords were dissected from 200 g rats, carefully cleaned fromadhering dorsal and ventral roots, and homogenized (polyton, 30 secondsat half maximal speed). Sciatic nerves were dissected, minced andhomogenized. Myelin fractions were isolated by flotation of low speedsupernatants on sucrose density gradients (Colman et al., 1982, J. CellBiol. 95:598-608). In some experiments, to remove possible trappedcontaminants, the crude membrane fraction was washed following hypotonicshock. Sedimentation in hypotonic medium was achieved at 10,000× g for 5minutes. Membrane fractions in sucrose solutions containing no more than50 mM ionic species were adsorbed for several hours onto the wells ofPLYS-coated tissue culture dishes (about 0.1 mg of protein per cm² oftissue culture dish). Unbound membranes were removed by three washeswith CMF-Hank's solution. Coated dishes were then immediately used insubstrate testing experiments. In experiments with sympathetic orsensory neurons small droplets of central or peripheral myelin weredeposited in defined patterns over 35 mm culture dishes.

Sympathetic or sensory neurons cultured as described above were examinedafter 12 hours to 4 days, neuroblastoma cells after 5-24 hours, and 3T3cells after 1-4 hours. For quantification, neuroblastoma cells wereclassified as round cells, cells with filopodia or short processes, orcells with processes longer than one cell body diameter. 3T3 cells wereclassified as round cells, cells with filopodia or short processes, orlarge flat cells. Three to four micrographs per culture were taken atrandom from 3 cultures for each experimental point.

6.2. RESULTS 6.2.1. CULTURES OF DISSOCIATED YOUNG OR ADULT RAT OPTICNERVES

GFAP positive astrocytes accounted for about 30% of the cells indissociated 10 day old rat optic nerves. About 50% of the cells werepositive for the O₄ antigen, a marker for differentiated,(GalC-positive) and immature (A₂ B₅ -positive) oligodendrocytes. Nooverlap was seen in the labeling between O₄ and GFAP or O₄ and Thy-1,confirming the specificity of the O₄ antibody as a marker for theoligodendrocyte family (Sommer and Schachner, 1981, Dev. Biol.83:311-327). Thy-1-positive fibroblasts with large flat morphologiesaccounted for about 20% of the cells in young rat optic nerves.

6.2.2. SUBTYPES OF OLIGODENDROCYTES

In cultures from 7-10 day old rats, about 50% of the O₄ -positive cellswere A₂ B₅ -labeled cells. Such cells were O₁ -negative (Table I) andhad different morphologies, including cells with irregular processesfrom polygonal cell bodies, flat cells with peripheral processes,bipolar cells, or cells decorated with filopodia. On the basis of thismarker profile (A₂ B₅ ⁺, O₄ ⁺, O₁ ⁻) and in agreement with Schnitzer andSchachner (1982, Cell Tissue Res. 2245:625-636), we interpret thesecells as being precursor and immature oligodendrocytes and collectivelycalled them "immature oligodendrocytes". This cell group is probablyheterogenous, as is also suggested by the different morphologies.Filopodia-carrying cells may be the most advanced (Table I).

                  TABLE I                                                         ______________________________________                                        A: OLIGODENDROCYTE SUBPOPULATIONS (7 DAY                                      OPTIC NERVES, 2 DAYS IN CULTURE) DIFFER                                       IN THEIR LABELING BY THE ANTIBODY A.sub.2 B.sub.5                                       Percentage of Labeled Cells                                                     A.sub.2 B.sub.5 +/O.sub.4.sup.-                                                          A.sub.2 B.sub.5 +/O.sub.4 +                                                              A.sub.2 B.sub.5.sup.- /O.sub.4 +            ______________________________________                                        Highly branched                                                                                                      91 ± 4. 4                           oligodendrocytes                                                              Cells with irregular                                                          or polygonal shapes                                                           flat membraneous cells                                                                        37 ± 4.sup.a                                                                           51 ± 6                                                                                12 ± 6                              process-bearing cells                                                                         18 ± 5                                                                                      74 ± 5                                                                            8 ± 2                              cells with filopodia                                                                              0                 43 ± 8                               ______________________________________                                        B: OLIGODENDROCYTE SUBPOPULATIONS (7-10 DAY                                   OPTIC NERVES, 2 DAYS IN CULTURE) CHARACTERIZED                                BY THE ANTIBODIES O.sub.1 (GalC) and A.sub.2 B.sub.5 *                                  Percentage of Labeled Cells                                                     A.sub.2 B.sub.5 +/O.sub.1.sup.-                                                          A.sub.2 B.sub.5 +/O.sub.1 +                                                              A.sub.2 B.sub.5.sup.- /O.sub.1 +            ______________________________________                                        Highly branched                                                                                                      93 ± 2. 2                           oligodendrocytes                                                              Cells with irregular                                                          or polygonal shapes                                                           flat membraneous cells                                                                       100          0          0                                      process-bearing cells                                                                         84 ± 6                                                                                      14 ± 6                                                                           1.5 ± 1.5                           cells with filopodia                                                                              91                      (8 ± 8)                        ______________________________________                                         *Dissociated 7-10 day old rat optic nerve cells were cultured on PORN for     2 days and labeled by either first antibody A.sub.2 B.sub.5 (detected by      antimouse FITC) followed by O.sub.4 or O.sub.1 (detected by                   antimouse-RITC) or vice versa. The proportion of doublelabeled cells was      calculated from the values obtained for A.sub.2 B.sub.5.sup.+                 /O.sub.4/1.sup.- and A.sub.2 B.sub.5.sup.+ /O.sub.4/1.sup.+  cells. Value     represent the means ± SEM of 4-6 cultures  #(120-200 cells/culture)        from 2 separate experiments.                                                  .sup.a This population of A.sub.2 B.sub.5.sup.+ /O.sub.4/1.sup.-  cells       contains type II astrocytes and precursor cells not expressing any            oligodendrocyte marker.                                                       .sup.b Variable, weak, granular staining                                 

About 50% of the O₄ -positive cells were A₂ B₅ -negative and O₁-positive after 2 days in culture under our culture conditions. Most ofthese cells showed a typical, highly branched radial process network.Due to this characteristic morphology we called these cells highlybranched oligodendrocytes (Table I). After 2 days in culture, mosthighly branched oligodendrocytes from optice neres of 10 day old ratswere stained with an antiserum against myelin basic protein (MBP). Wetherefore interprete these cells as being myelin formingoligodendrocytes. Their characteristic process network may be the resultof an unstable, partially collapsed myelin membrane containingoccasional flat membrane areas. The total yield of cells from adultnerves was very low. Both, differentiated O₁ -positive highly branchedoligodendrocytes as well as immature A₂ B₅ -positive oligodendrocyteswere also present in cultures of adult tissue.

6.2.3. RESPONSE OF VARIOUS CELL TYPES TO HIGHLY BRANCHEDOLIGODENDROCYTES 6.2.3.1. CO-CULTURES WITH SYMPATHETIC OR SENSORYNEURONS

Dissociated cells from newborn rat superior cervical ganglia or dorsalroot ganglia were added to glial cells after 2-10 days in culture.Ganglionic Schwann cells and fibroblasts were eliminated by pulses ofAra C in some of the experiments. NGF (50 or 100 ng/ml) was added to theculture medium, leading to a rapid fiber outgrowth and to the formationof dense neurite networks within a few days. NGF alone had no effect onthe occurrence and morphology of oligodendrocytes. Glial cell types wereidentified by antibody staining at the end of the experiments (2 days to2 weeks of co-culture).

In cultures with a dense neurite plexus, the most striking observationwas the occurrence of "windows" free of neurites in the center of whichcells with radial, highly branched processes could be observed (FIGS.1A-H). Antibody staining identified these cells as highly branchedoligodendrocytes. A quantification of the interaction ofoligodendrocytes with sympathetic ganglion cells is shown in FIGS. 2Aand 2B. Astrocytes adjacent to oligodendrocytes were rare in thesecultures since the overall glial cell density was low; preferentialassociation with astrocytes could, therefore, not account for thisresult. Highly branched oligodendrocytes excluded neurons from theirterritory irrespective of the culture substrate used. The same "windows"were formed on plain plastic, collagen, PORN- or laminin-coated culturedishes. No difference was seen between sympathetic and sensory neurons;both were excluded from the territory of highly branchedoligodendrocytes. Likewise, Schwann cells, when present, did not invadeor overgrow the oligodendrocyte process networks (FIG. 1B). In contrast,immature oligodendrocytes, characterized by their irregular shapes andthe absence of O₁ -antigen, did allow neurite growth on their processesand cell bodies (FIGS. 1B, 1E, 1F). A₂ B₅ could not be used as a markerfor immature oligodendrocytes in co-cultures with neurons, as thisantigen was rapidly lost after addition of the neurons. Recent directobservations of the encounter of growth cones with oligodendrocytesshowed that growth cone movement was arrested after filopodial contactis established. Normal growth cone activity was seen during contact andcrossing of immature cells. These observations also exclude thepossibility that the "windows" were formed secondarily in the neuriteplexus. Astrocytes in the same cultures were often overgrown by singleneurites or neurite bundles (FIGS. 3A, 3B). This was true for bothmorphological types, flat and stellate cells.

6.2.3.2. CO-CULTURES WITH FETAL RAT RETINAL CELLS

After plating retinal cells at monolayer density on top of 5 day oldcultures of optic nerve non-neuronal cells, a typical rearrangement ofthe retinal cells could be observed; whereas oligodendrocyte precursorcells were often contacted by retina cells, the highly branchedoligodendrocytes were mostly free of them (FIGS. 1G, 1H, 3C, 3D). Again,astrocytes were preferred as a substrate over PORN.

6.2.3.3. RESPONSE OF OTHER CELL TYPES TO HIGHLY BRANCHEDOLIGODENDROCYTES

Neuroblastoma cells (line NB-2A) were plated at high cell density intodissociated optic nerve cultures and stimulated for fiber production by2 mM dibutyryl-cyclic-AMP or by GdNPF. Seven, 24 or 48 hours later, thecultures were fixed and oligodendrocytes were identified by antibodiesO₄ and ₁. Again, the territories of highly branched oligodendrocyteswere clearly spared by neuroblastoma cells (FIGS. 4A, 4B). Processesproduced by neuroblastoma cells situated close to oligodendrocytes werepointing away from the oligodendrocytes (FIGS. 4A, 4B; FIG. 5 and TableIA).

                  TABLE IA                                                        ______________________________________                                        ORIENTATION OF NEUROBLASTOMA PROCESSES                                        WITH REGARD TO HIGHLY BRANCHED OLIGODENDROCYTES                                         % of Processes in Each Sector                                                   Adjacent Neuro-                                                                           Distant Neuro-                                        Sector†                                                                            blastoma Cells                                                                            blastoma Cells                                        ______________________________________                                        1            7 ± 1.4 25 ± 2.4 ***                                       2           34 ± 1.2 26 ± 1.2 ***                                       3           33 ± 2.7 25 ± 2.3 *                                         4           26 ± 2.3 24 ± 2.7                                           ______________________________________                                         †Shown in FIG. 5                                                       * p < 0.0.05                                                                  *** p < 0.001                                                            

Primary culture fibroblasts and astrocytes in the optic nervepreparations as well as mouse 3T3 cells showed a drastic avoidancebehaviors towards highly branched oligodendrocytes. 3T3 cells plated athigh cell density into optic nerve glial cultures attached and flattenedout between 30 minutes and 3 hours on the PORN substrate. In theseforming monolayers, characteristic "windows" appeared corresponding tothe territories of highly branched oligodendrocytes (FIGS. 4C, 4D). Atthe sites of contact, 3T3 cells formed a crescent-shaped bulge ofcytoplasm. Lamellipodia were absent in this region. Significantly,fibroblasts that landed directly on highly branched oligodendrocytescompletely failed to spread. As for neurons, immature oligodendrocyteswere not visibly avoided by 3T3 cells (FIGS. 6A-B).

6.2.4. ABSENCE OF SPECIES SPECIFICITY

Neither the specific morphology nor the unfavorable substrate propertyof oligodendrocytes were species specific. Dissociated non-neuronalcells from E13 and E17 chick optic nerve contained besides O₄-positive/A₂ B₅ -negative/O₁ -positive highly branched oligodendrocytes.3T3 cells plated on top of chicken non-neuronal cells formed thecharacteristic "windows" around these chick oligodendrocytes.

6.2.5. MYELIN AS A SUBSTRATE

The properties of myelin as a substrate for neurons or fibroblasts werealso tested, since myelin consists of spirally wrapped oligodendrocytemembranes. Crude myelin fractions from adult rat spinal cord or sciaticnerve were prepared by flotation on a sucrose gradient. The myelin wasadsorbed to PLYS-coated tissue culture dishes and tested for itssubstrate properties for superior cervical ganglion cells, dorsal rootganglion cells, neuroblastoma cells and 3T3 cells. All four cell typesattached poorly to CNS myelin and showed marked difficulties in theirprocess outgrowth. Sympathetic and sensory neurons on CNS myelinremained round or produced short, abortive fibers in spite of thepresence of NGF (50 ng/ml or 100 ng/ml) (FIGS. 7A, l7C). In contrast,long fibers were produced on islets of sciatic nerve myelin in the sameculture dishes (FIGS. 7B, l7D). Small CNS myelin islets on PLYS appearedas "windows" outlined by excluded neurites, whereas PNS myelin-PLYSboundaries were apparently not detected by growing neurites.

Process outgrowth from neuroblastoma cells (line NB-2A) in the presenceof dibutyryl-cyclic AMP was significantly reduced by CNS myelin (FIG.8A).

Spreading of 3T3 fibroblasts was strongly inhibited by CNS myelin (FIG.8B). 3T3 cells remained round or produced spindle-shaped or polygonalmorphologies with a minimal cell substrate interaction. In contrast,large flat membranes were produced within 20-30 minutes on polylysineand, with a somewhat slower time-course, also on myelin from theperipheral nervous system (FIG. 8B). Nonpermissiveness was associated,at least in large part, with myelin membranes, since sedimentation at10,000× g for 5 minutes under hypotonic conditions (see Section 6.1.5.,supra) was sufficient to pellet most nonpermissive membranes. Underthese conditions, most surface membrane components floating to densitiessmaller than the one of 0.85 M sucrose, would not be expected tosediment.

CNS myelin nonpermissiveness is not due to astrocyte membranes, since acell membrane preparation from CNS tissue containing minimal amounts ofwhite matter (superficial cortical layers) was a permissive substratefor fibroblast spreading.

These experiments show that, in parallel to the effects of living,highly branched oligodendrocytes, myelin from the CNS is also a stronglynonpermissive substrate for primary culture neurons, neuroblastomacells, and 3T3 fibroblasts. Myelin from the peripheral nervous systemdoes not show a comparable nonpermissive substrate effect.

6.3. DISCUSSION

In the present study, we observed that myelin forming oligodendrocytesand isolated CNS myelin exert a nonpermissive substrate effect onoutgrowing neurites of sympathetic and sensory neurons and neuroblastomacells, as well as for the attachment of retinal cells and the spreadingof fibroblasts.

Several classes of cells were present in short-term cultures ofdissociated rat optic nerves: oligodendrocytes, astrocytes(GFAP-positive) fibroblasts (Thy-1, fibronectin-positive) and severaltypes of precursor cells. Within the oligodendrocyte family (O₄-positive; Sommer and Schachner, 1981, Dev. Biol. 83:311-327), one mainsubtype of cells was characterized by the absence of the O₁ antigen(GalC) and of MBP, two components highly characteristic of myelin(Mirsky, et al., 1980, J. Cell Biol. 84:483-494), and the presence ofbinding sites for the antibody A₂ B₅. A₂ B₅ was shown to be a marker foroligodendrocyte/type II astrocyte precursors, type II astrocytes, andneurons (Schnitzer and Schachner, 1982, Cell Tissue Res. 224:625-636;Abney, E. R. et al., 1981, Dev. Biol. 100:166-171; Raff, et al., 1983,Nature 303:390-396). Therefore, we considered this cell class torepresent immature oligodendrocytes, probably including precursors suchas those described by Dubois-Dalcq (1986, Soc. Neurosci. Abstr. 12:767)and Sommer and Noble (1986, Soc. Neurosci. Abstr. 12:1585). The presenceof O₄ distinguishes these cells from the O2A precursors (Raff, M. C. etal., 1983, Nature 303:390-396). These immature cells showed irregularand variable morphologies with bipolar shapes or polygonal cell bodiesand irregular processes, often decorated with filopodia. The cell classis probably heterogenous; cell division could be observed. The secondmain oligodendrocyte subclass consisted of A₂ B₅ -negative, O₁ -positivecells, possessing a radial, highly branched and anastomosing processnetwork. Most of these highly branched oligodendrocytes in 2 day oldcultures of 10 day old rat optic nerves were positive for MBP under ourculture conditions. We thus interpret this frequent cell type asrepresenting oligodendrocytes actively involved in the synthesis ofmyelin membranes which are deposited flat on the culture substrate inthe absence of axons. These membranes are unstable and collapse to formthe characteristic, anastomosing process network. This cell type hasbeen described as "hairy eyeball cell" (Sommer and Schachner, 1981, Dev.Biol. 83: 311-327), and formation of whorls of compact myelin by suchcells has been observed after prolonged times in culture (Rome et al.,1986, J. Neurosci. Res. 15:49-65; Yim et al., 1986, J. Biol. Chem.261:11808-11815).

Both immature and myelin forming oligodendrocytes were seen in culturesof 7 to 10 day old or adult rat optic nerves, and also in cultures of 1day rat optic nerves, newborn rat spinal cord or adult rat corpuscallosum, as well as in cultures of spinal cord and optic nerves of E13or E17 chicken embryos. Immature cells clearly were predominant indissociates from younger stages, but the large drop in cell yield upondissociation with increasing age precluded any quantitative populationanalysis. However, immature oligodendrocytes could also be obtainedconsistently from adult rat white matter tissues, confirming earlierobservations by French-Constant and Raff (1986, Nature 319:499-502).

The addition of neurons to established glial cultures showed dramaticdifferences in substrate properties for neuronal attachment and fiberoutgrowth among the various types of non-neuronal cells. Astrocytes,particularly the flat reactive protoplasmic astrocytes, were adhesiveand favorable for neuronal attachment and outgrowth, in agreement withearlier observations (Foucaud et al., 1982, Cell Res. 137:285-294;Hatten, et al., 1984, J. Cell Biol. 98:193-204; Noble, et al., 1984, J.Neurosci. 4:1982-1903; Fallon, 1985, J. Cell Biol. 100:198-207).Immature oligodendrocytes also were frequently contacted by neurites ornerve cell bodies. This behavior could be of high physiologicalrelevance. During development, oligodendrocyte precursors migrate intothe already formed axonal bundles and extend processes to contact acertain number of axons. These processes then start to enwrap and spiralaround the axons, thus forming the structure called myelin (Wood andBunge, 1984, W. T. Norton, ed., 1-46).

In sharp contrast to astrocytes and oligodendrocyte precursors, we foundthat myelin forming oligodendrocytes display strongly nonpermissivesubstrate properties for neuronal attachment and fiber outgrowth as wellas for fibroblast attachment and spreading. This effect was strong andpronounced even on laminin-coated culture dishes, which otherwiserepresent an excellent substrate for neurite growth (Manthorpe, et al.,1983, J. Cell Biol. 97:1882-1980; Rogers, et al., 1983, Dev. Biol.98:212-220). This effect was not overcome by high doses of NGF incultures of sympathetic and sensory neurons, or GdNPF ordibutyryl-cyclic-AMP in cultures of neuroblastoma cells. A similar oridentical nonpermissive substrate property was associated with rat CNSmyelin but not with myelin from peripheral nerves. The effect wasstrictly contact-dependent, since nerve cells or fibroblasts grew welland were free to move in the immediate surrounding of theseoligodendrocytes or of CNS myelin islets. Mouse 3T3 cells were alsoinhibited by chicken oligodendrocytes, showing that this effect is notspecies-specific.

In the rat optic nerve, the peak number of axons is reached at embryonicday 20, followed by a dramatic loss of axons (Crespo, et al., 1985, Dev.Brain Res. 19:129-134). Oligodendrocyte precursors appear from E17onward (Raff, et al., 1985, Cell 42:61-69) and express GalC around birth(Miller, et al., 1985, Dev. Biol. 111:35-41). The first myelindetectable by electron microscopy appears at postnatal day 6 (Hildebrandand Waxman, 1984, J. Comp. Neurol. 224:25-37). This clear-cut temporaldissociation between axonal growth and myelin formation is also presentin chicken optic nerves (Rager, 1980, Cell Biol. 63:1-92) and, althoughless well studied, in many white matter tracts of the CNS (Matthews andDuncan, 1971, J. Comp. Neurol. 142:1-22; Looney and Elberger, 1986, J.Comp. Neurol. 248:336-347). During normal development, growing axonstherefore probably never encounter myelin or myelinatingoligodendrocytes within their fascicles, but rather interact withprecursors and immature oligodendrocytes. The extremely slow time-courseobserved for in vitro myelination (Wood, et al., 1980, Brain Res.196:247-252; Wood and Williams, 1984, Dev. Brain Res. 12:225-241) couldbe consistent with a situation where undifferentiated oligodendrocytesfirst interact with axons and are then induced to differentiate and toform myelin.

In contrast to development, during CNS regeneration, axonal growth conesor sprouts do encounter mature oligodendrocytes and myelin. Substrateproperties of CNS tissue, in particular the absence of potent neuritepromoting substrates like laminin in the differentiated CNS of highervertebrates, are important aspects in the context of CNS regeneration(Liesi, 1985, EMBO J. 4:2505-2511; Carbonetto, et al., 1987, J.Neurosci. 7:610-620). However, since myelin and oligodendrocytes persistfor a long time in denervated CNS tracts (Fulcrand and Privat, 1977, J.Comp. Neur. 176:189-224; Bignami, et al., 1981, J. Neuropath, Exp.Neurol. 40:537-550), the absence of any fiber regeneration in whitematter areas in contrast to peripheral nerves and PNS/CNS transplantscould be related to these nonpermissive substrate factors.

Under normal conditions, blocking certain territories for later growingaxonal populations during development, antagonism between favorable andnonpermissive substrate molecules during development of CNS projectionpatterns, or the spatial limitation of sprouting in the differentiatedCNS are possible functions for this oligodendrocyte associatednonpermissive substrate property.

7. TWO MEMBRANE PROTEIN FRACTIONS FROM CENTRAL NERVOUS SYSTEM MYELINWITH INHIBITORY PROPERTIES FOR NEURITE GROWTH AND FIBROBLAST SPREADING

We have searched for surface components in CNS white matter, which wouldprevent neurite growth. CNS, but not PNS, myelin fractions from rat andchick were highly nonpermissive substrates in vitro. We have used an invitro spreading assay with 3T3 cells to quantify substrate qualities ofmembrane fractions and of isolated membrane proteins reconstituted inartificial lipid vesicles. CNS myelin nonpermissiveness was abolished bytreatment with proteases and was not associated with myelin lipid.Nonpermissive proteins were found to be membrane bound and yieldedhighly nonpermissive substrates upon reconstitution into liposomes. Sizefractionation of myelin protein by SDS-PAGE revealed two highlynonpermissive minor protein fractions of molecular weight, 35 kD and 250kD. Removal of kD and of 250 kD protein fractions yielded a CNS myelinprotein fraction with permissive substrate properties. Supplementationof permissive membrane protein fractions (PNS, liver) with low amountsof 35 or of 250 kD CNS myelin protein was sufficient to generate highlynonpermissive substrates. Inhibitory 35 and 250 kD proteins were foundto be enriched in CNS white matter and were found in optic nerve cellcultures which contained highly nonpermissive, differentiatedoligodendrocytes.

The data presented herein (Caroni and Schwab, 1988, J. Cell Biol.106:1281-1288) demonstrate the existence of 35 kD and 250 kD myelinmembrane-bound proteins with potent nonpermissive substrate properties.Their distribution and properties suggest that these proteins might playa crucial inhibitory role during development and regeneration in CNSwhite matter.

7.1. MATERIALS AND METHODS 7.1.1. CELL CULTURE

Mouse NIH 3T3 cells were cultured and assayed for spreading behavior inDMEM containing 10% FCS. In control experiments, use of definedserum-free medium did not alter responses of 3T3 cells to testedsubstrates. Mouse neuroblastoma cells (line NB-2A) were cultured in DMEMwith 10% FCS in the presence of either 1 mM dibutyryl-cAMP or ofglia-derived neurite promoting factor (GdNPF). Superior cervical anddorsal root ganglia from newborn rats were dissected and dissociatedinto single cells as described (Mains and Patterson, 1973, J. Cell Biol.59:329-345; Schwab and Thoenen, 1985, J. Neurosci. 5:2415-2423). Neuronswere cultured in an enriched L15 medium with 5% rat serum (Mains andPatterson, 1973, J. Cell Biol. 59:329-345) and with 100 ng/ml of 2.5Snerve growth factor. Overgrowth by contaminating dividing cells wasprevented by inclusion of cytosine arabinoside (10⁻⁵ M) in the culturemedium.

7.1.2. SOURCES OF TESTED SUBSTRATES

Myelin fractions were all prepared by the same procedure, involvingtissue homogenization in isotonic sucrose buffer with a polytron (modelPCU-2; Kinematica, Luzern, Switzerland) homogenizer (setting 4, twotimes 30 s, on ice) and flotation of a low speed supernatant onto adiscontinuous sucrose gradient (myelin collected at 0.25 M sucrose toplayer) (Colman, et al., 1982, J. Cell Biol. 95:598-608). All isolationmedia contained trasylol (aprotinin; Sigma Chemical Co., St. Louis, Mo.;100 U/ml), 5 mM iodoacetamide, and 5 mM EDTA to reduce proteasedigestion (this reagent mixture is designated below as proteaseinhibitors). Finally, myelin fractions were washed hypotonically in 30mM Hepes (pH 7.4) (medium A) plus protease inhibitors and frozen inaliquots at -80° C. CNS myelin fractions were prepared from spinal cordscarefully stripped of ventral and dorsal roots, or from rat opticnerves. The following sources were used: rat, spinal cord, 3 month oldLewis rats, male; chick, spinal cord, P21; trout, spinal cord, 2 year;frog, spinal cord, 6 months. PNS myelin fractions were prepared from ratsciatic nerves (3 month male). Rat liver cell membranes were prepared bystandard procedures, involving mild isotonic homogenization andcollection of membranes at the density of 0.25 M sucrose (discontinuoussucrose gradient). Rat CNS tissue enriched in gray matter was obtainedfrom superficial neocortex layers, whereas white matter-enriched tissueconsisted of the corpus callosum.

7.1.3. SUBSTRATE-ASSAYING PROCEDURE

Substrates to be tested, in 40-70 mOsmol solutions, were dried ontopolylysine (PLYS)-coated tissue culture dishes. Unbound membranes andsolutes were removed by three washes with Ca² + Mg²⁺ -free Hank'ssolution. Coated dishes were then immediately used in substrate-testingassays. For most experiments, substrates were dried onto the wells ofdishes (35-mm dishes with four internal wells; Greiner, Nurtingen,Federal Republic of Germany). 3T3 cells were detached from ˜30%confluent cultures by brief trypsin (0.2%) treatment in 37° C. PBS plusEDTA. Trypsinization was stopped by 10-fold excess of serum-containingDMEM; cells were collected and resuspended in DMEM-10% FCS atappropriate concentrations. 30,000 cells/cm² were added to precoatedculture dishes, and experiments were scored after 1 hour in culture. Insome cases, due to occasional slower spreading behavior of 3T3 cellpopulations, scoring had to be delayed up to 2 hours in culture. Afterperiods of more than ˜5 hours, inhibitory properties of myelin fractionsin the absence of serum were less pronounced than observed in thepresence of 10% FCS, possibly due to the presence of substrate digestingproteases. If substantial spreading on PLYS-coated dishes was notobtained within 2 hours in culture, tests were discarded and repeatedwith a fresh batch of cells. Quantitative evaluation of spreading wasperformed with a surface integration program on at least 30 cells perexperimental point. Only spread cells were considered and azero-spreading value (strongly refractory and round cells) wassubtracted. Each experiment was repeated at least five times.Experiments were found to be subject to only small quantitativevariations, and values from representative experiments are given.Spreading degrees are given as means ±standard error of the mean. Whenrecoveries of inhibitory activity were estimated, serial dilutions ofliposomes (in medium A) were assayed for nonpermissiveness. In somecases, in order to detect differences among strong inhibitorysubstrates, 3T3-spreading times were extended to up to 5 hours. Recoveryvalues are based on internal calibration with a CNS myelin liposomestandard, and are to be considered as first approximations. When neuriteextension was evaluated, neuroblastoma cells or superior cervicalganglia neurons were seeded at ˜25,000 cells/cm² and experiments werescored after 24 hours in culture.

7.1.4. SUBSTRATE PROCESSING

Protease sensitivity of inhibitory fractions was determined by digestingwashed, protease inhibitor-depleted membranes with trypsin. Membranefractions (concentrations of maximally 1 mg of protein per ml) wereexposed for 10 minutes at room temperature to 0.1% trypsin. Digestionwas interrupted by the addition of 0.2% trypsin inhibitor (sigmaChemical Co.) and membranes were either washed in medium A or separatedfrom protease by Sephadex G-50 chromatography (liposomes). Under theseconditions, trypsin was retarded by the column, whereas liposomes wererecovered in the excluded volume. Digested, washed membranes werefinally adsorbed to culture dishes, and their substrate properties wereanalyzed as described supra. In some experiments with myelin fractions,pronase (Sigma Chemical Co.) or elastase (Sigma Chemical Co.) were used.In those instances, protease was removed by three washes of the myelinin 30 mM Hepes, pH 7.4.

Extraction of peripheral membrane proteins from CNS myelin was performedby resuspending membranes in either 4 M guanidinium chloride (Merck &Co., Inc. Rahway, N.J.)/30 mM Hepes or in 500 mM unbuffered Tris base(Sigma Chemical Co.). Protease inhibitors were routinely included in theextraction buffers. After incubation for 30 minutes at room temperature,myelin was sedimented, washed in medium A, and assayed for its substrateproperties.

Ethanol/ether (2:3 vol ratios) extraction of myelin was performed by astandard procedure (see, for example, Everly, J. L. et al., 1973, J.Neurochem. 21:329-334). Solvent-insoluble fraction was reconstitutedinto lipid vesicles (see Section 7.1.5,) infra). The lipid-containingsoluble fraction was dried and reconstituted by the cholate method (seeSection 7.1.5, infra).

7.1.5. LIPOSOMES

Liposomes were prepared in medium A by the cholate method (Brunner, etal., 1978, J. Biol. Chem. 253:7538-7546). Protein was solubilized in 2%SDS (in medium A plus protease inhibitors); insoluble protein wassedimented and discarded. Solubilized protein was precipitated with a30-fold excess of acetone. To obtain reproducible yields, acetoneprecipitation was allowed to proceed for 15 hours at 4° C. Proteinextracts from tissues were prepared by homogenization of minced tissuewith a glass-teflon potter in 2% SDS-containing, proteaseinhibitors-supplemented, medium A. Solubilized protein was thenprecipitated with ice-cold acetone as described above. Extracts fromcultured cells were prepared by, first, detaching the cells with arubber policeman in the presence of PBS plus EDTA plus proteaseinhibitors, and by then homogenizing suspended cells with a glass-teflonpotter. Upon low-speed pelleting of nuclear material, 2% SDS was addedto supernatants and solubilized protein was precipitated with ice-coldacetone. In all cases, acetone-precipitated protein was sedimented(10,000× g, 15 minutes) and resuspended at 1 mg/ml in medium A with 2.5%cholate. Phospholipids (phosphatidylcholine/phosphatidylserine, 10:1)dissolved in medium A with 2.5% cholate were then added (˜5-10:1 ratioof added phospholipid to protein) and liposomes were formed on aSephadex G-50 column. When gel-extracted protein was reconstituted,precipitated protein was resuspended at ˜50 μg/ml and phospholipid toprotein ratios were up to 100:1.

A number of control experiments were performed. Thus, acetoneprecipitated myelin or gel-extracted protein did not prevent 3T3spreading when resuspended in medium A or in medium A with 2.5% cholateand adsorbed directly onto PLYS-coated dishes. Also, running ofcholate-solubilized protein on the Sephadex G-50 column, in the absenceof phospholipids did not yield nonpermissive fractions. Experiments with¹²⁵ I-labeled CNS myelin protein showed that ˜30% of applied label wasrecovered in the liposome fraction from the G-50 column. In someexperiments, lipid vesicles were formed in the presence of trace amountsof [³ H] cholesterol and were then dried onto the wells of tissueculture dishes (Greiner). Total culture dish associated membrane amounts([³ H] cholesterol) were found to vary independently of tested protein,indicating that differences in liposome binding to culture dishes cannotbe responsible for the observed differences in substrate properties.

7.1.6. GEL-EXTRACTED PROTEIN FRACTIONS AS SUBSTRATE

Protein was run on 3-15% gradient gels under reducing conditions. Forthis purpose, samples were preincubated for 30 minutes at roomtemperature in sample buffer containing 2% SDS and β-mercaptoethanol.Thin lanes were cut and stained with Coomassie Brilliant Blue or withthe silver method. Protein bands to be analyzed were carefully alignedwith the unstained gel parts to be extracted. Gel regions from theunstained gel part were cut, and minced gel was extracted for 1 hourwith 0.5% SDS. In most experiments, 50 μg/ml of insulin (Sigma ChemicalCo.) were included in order to reduce losses due to adsorption ofprotein present in low concentration. Insulin was selected for itspurity and for its small size, resulting in efficient separation fromthe liposome fraction. In control experiments, no substrate differencescould be detected when 50 μg/ml of insulin were added to variousreconstitution mixtures, including protein-free liposomes. Gel-extractedprotein was precipitated with a 10-fold excess of ice-cold acetone (15hours), and sedimented protein was resuspended in cholate buffer.Protein was stored frozen in cholate buffer and reconstitution mixtureswere prepared from these protein stocks. Reconstitution and test ofsubstrate properties were performed as described above.

The amount of protein present was determined by the filter bindingmethod (Schaffner and Weissman, 1973, Anal. Biochem. 56:502-514) withBSA (Sigma Chemical Co.) as a standard.

7.2. RESULTS 7.2.1. NONPERMISSIVE SUBSTRATE EFFECT IS FOUND IN CNSMYELIN OF HIGHER VERTEBRATES (CHICK, RAT), BUT NOT OF LOWER VERTEBRATES(TROUT, FROG)

Rat CNS myelin was found to be a nonpermissive substrate for neuriteoutgrowth from rat superior cervical ganglion neurons and for spreadingand migration of 3T3 fibroblasts (see also section 6, supra). Analogousresults were obtained when myelin was prepared from rat optic nerve orfrom rat brain. Neuron type apparently did not influence substrateresponse as similar results were obtained with dorsal root ganglionneurons. Likewise, rat CNS myelin nonpermissiveness was observed fordibutyryl-AMP-induced or GdNPF-induced outgrowth from neuroblastomacells. Thus, nonpermissiveness of rat CNS myelin is apparently generalwith regard to neuron type and induction of neurite outgrowth.

Lack of regenerative fiber growth is found in the CNS of highervertebrates but not in those of fishes and to a limited extent in thoseof amphibia (Bohn, et al., 1982, Am. J. Anat. 165:307-419; Stensas,1983, In Spinal Cord Reconstruction, C. C. Kao, R. P. Bunge, and P. J.Reier, eds., Raven Press, New York 121-149; Hopkins, et al., 1985, J.Neurosci. 5:3030-3038; Liuzzi and Lasek, 1986, J. Comp. Neurol.247:111-122). We prepared spinal cord myelin fractions from trout, frog,and chick in order to determine potential differences in substrateproperties. Trout (FIG. 9) and frog (FIG. 9) CNS myelin fractions werefound to have substrate properties similar to those of rat PNS myelin,whereas CNS myelin from the chick (spinal cord, postnatal 21) was anonpermissive substrate, although slightly less so than its ratcounterpart.

7.2.2. MEMBRANE-BOUND PROTEIN FRACTION OF RAT CNS MYELIN IS RESPONSIBLEFOR ITS NONPERMISSIVE SUBSTRATE PROPERTIES

Rat CNS myelin was processed by standard procedures in order todetermine the nature of the component(s) responsible for itsnonpermissive substrate properties. Fractions were tested for reductionof 3T3 fibroblast spreading. Data are shown in Table II.

                  TABLE II                                                        ______________________________________                                        NONPERMISSIVENESS OF CNS MYELIN                                               IS DUE TO MEMBRANE-BOUND PROTEIN*                                                                  3T3 spreading                                            Substrate            (μm.sup.2)                                            ______________________________________                                        Tissue culture plastic                                                                             1,646 ± 309                                           CNS myelin                                                                    Untreated              211 ± 30                                            Trypsin-treated      1,344 ± 181                                           Liposomes                                                                     Ethanol/ether-soluble myelin fraction                                                              1,253 ± 159                                           Ethanol/ether-insoluble myelin fraction                                                              226 ± 45                                            Artificial lipid vesicles, no additions                                                            1,328 ± 136                                           ______________________________________                                         *Spreading extent of 3T3 cells was estimated after 1 hour in culture.         Protein amounts to be adsorbed to wells of dishes (Grenier) were 20 μg     of CNS myelin protein per cm.sup.2. In the solvent extraction experiments     100 μg of CNS myelin protein were extracted and onefifth of resulting      liposomecontaining volume was dried onto wells. These myelin quantities       represent about 10 times saturation levels with respect to observed           nonpermissiveness.                                                       

Brief treatment of the myelin with trypsin abolished nonpermissiveness.Similar results were obtained with elastase or with pronase treatment.

Extraction of the myelin under conditions that solubilize peripheralmembrane proteins (4 M guanidinium chloride or pH 10.5) failed todissociate nonpermissiveness from low speed myelin membrane pellets.Lipid extraction with ethanol/ether yielded a permissive lipid fractionand a nonpermissive protein fraction (Table II). The latter requireddetergent to be solubilized and had to be incorporated into lipidvesicles in order to permit detection of nonpermissive substrateproperty. In control experiments, phosphatidylcholine/phosphatidylserineliposomes were a slightly less favorable substrate than tissue cultureplastic (Table II). When CNS myelin protein-containing liposomes weresubjected to trypsin treatment, their nonpermissive substrate propertieswere abolished (Table III). Thus, a membrane-bound protein fraction fromrat CNS myelin is a nonpermissive substrate for 3T3 fibroblastspreading. That fraction can apparently be reconstituted in active forminto artificial lipid vesicles. In control experiments, protein frommembrane fractions with permissive substrate properties yielded, uponreconstitution, liposomes that were permissive for 3T3 spreading (TableIII).

                  TABLE III                                                       ______________________________________                                        NONPERMISSIVE SUBSTRATE PROPERTY                                              OF CNS MYELIN IS PRESERVED UPON                                               RECONSTITUTION INTO ARTIFICIAL LIPID VESICLES                                                           Dish-Adsorbed                                       Reconstituted  3T3 Spreading                                                                            Lipids (cpm                                         Protein Fraction                                                                             (μm.sup.2)                                                                            [.sup.3 H]cholesterol)                              ______________________________________                                        No protein     1,638 ± 91                                                                            521 ± 65                                         CNS myelin      136 ± 30                                                                             650 ± 58                                         CNS myelin; resulting                                                                        1,397 ± 152                                                                           630 ± 32                                         liposomes trypsinized                                                         PNS myelin     1,570 ± 136                                                                           620 ± 41                                         Liver membranes                                                                              1,445 ± 121                                                                           750 ± 47                                         ______________________________________                                         *Tested protein fractions (100 μg) were reconstituted and onefifth of      resulting liposomecontaining volume (60 μl) was absorbed to wells. The     adsorbed volume contained 20,000 cpm [.sup.3 H]cholesterol. Dishadsorbed      counts were determined upon SDS solubilization of adsorbed liposomes. For     these experiments, liposomes were removed prior to fibroblast addition.       Trypsinisation of CNS myelin liposomes and separation of inhibitorblocked     trypsin  #  from vesicles was performed as described in Section 7.1.4,        supra.                                                                   

7.2.3. IDENTIFICATION OF 35 kD AND 250 kD MINOR PROTEINS FROM MYELIN ASNONPERMISSIVE SUBSTRATES FOR FIBROBLAST SPREADING AND NEURITE OUTGROWTH

As myelin nonpermissiveness partially survived denaturing procedures,attempts were made to identify responsible components followingseparation by SDS-PAGE. In preliminary experiments, it was found thatsolubilization of myelin proteins in SDS-PAGE sample buffer followed byreconstitution of acetone-precipitated protein yielded a fractionpossessing )30% of starting nonpermissiveness. Apparent activityrecoveries were estimated by assaying serial dilutions of reconstitutedprotein with the 3T3 fibroblast spreading assay. As a comparison,solubilization in 2% NP-40™, 0.5% Na-deoxycholate yielded apparentactivity recoveries of )80%. When CNS myelin protein was run on SDS-PAGEand the entire gel was then extracted with 0.5% SDS, recoveries ofnonpermissive substrate activity were )20%. Activity could be recoveredin approximately equal amounts ()10% of applied activity) from gelregions corresponding to the migration distance of 35 kD and of 250 kDproteins, respectively (FIG. 10). The inhibitory proteins were highlyeffective, as just 10 ng of 250 kD protein per cm² of culture dish wasrequired to obtain half-maximal inhibition. Neither the 250 kD nor the35 kD region contained major myelin protein bands (each region contained)3% of total silver stained myelin protein). These gel regionsapparently contained more than one protein species. Reconstitution ofpooled gel regions depleted of kD and of 250 kD proteins yieldedpermissive liposomes (FIGS. 11A-C). Thus, 35 kD and 250 kD proteinsaccount for most of the nonpermissive substrate activity ofgel-extracted CNS myelin protein. Similarly to unfractionated myelin, 35kD and 250 kD proteins were nonpermissive substrates for fibroblastspreading and for neurite extension (FIGS. 11A-C). In controlexpriments, sciatic nerve protein or a liver homogenate did not generate250 kD nor 35 kD nonpermissive protein fractions (FIG. 12). It seems,therefore, reasonable to conclude that the protein fractions identifiedabove are responsible for the marked nonpermissive substrate propertiesof rat CNS myelin in vitro.

We next asked whether addition of these proteins to fractions withneutral substrate properties is sufficient to generate a nonpermissivesubstrate. As shown in FIG. 12, liver protein and sciatic nerve proteincould yield nonpermissive substrates for 3T3 cells when supplementedwith 250 kD or with 35 kD proteins from rat CNS myelin as shown in FIG.12. In these experiments, 250 kD and 35 kD proteins were added toamounts of liver (or sciatic nerve) protein equivalent to the ones oftotal CNS myelin protein from which they were prepared. We conclude that35 kD and 250 kD proteins from rat CNS myelin act as inhibitors ofneurite outgrowth and of fibroblast spreading, as their additionconverts a neutral substrate into a nonpermissive one.

7.2.4. NONPERMISSIVE SUBSTRATE PROPERTY IS ENRICHED IN CNS WHITE MATTERAND IN CULTURED OLIGODENDROCYTES

Considering the documented poor regenerative fiber growth found inmature CNS white matter (Nornes, H. A., et al., 1983, Cell Tissue Res.230:15-35; Bjorklund, A. and Stenevi, U., 1984, Annu. Rev. Neurosci.7:279-308), it was of particular interest to determine whether the 35 kDand 250 kD neurite outgrowth-inhibiting proteins from CNS myelin wereenriched in CNS white matter and in myelin forming cells.Protein-containing lipid vesicles from homogenates of different CNSregions were prepared and their substrate properties were determined.Rat CNS white matter material yielded highly nonpermissive liposomescontaining inhibitory 250 kD and 35 kD protein fractions (Table IV).

                  TABLE IV                                                        ______________________________________                                        DISTRIBUTION OF INHIBITORY 250 kD AND 35 kD                                   PROTEIN FRACTIONS                                                                      3T3 Spreading on Liposomes From:                                                            250 kD    35 kD                                                   Total Protein                                                                             Fraction  Fraction                                     Protein Source                                                                           (μm.sup.2)                                                                             (μm.sup.2)                                                                           (μm.sup.2)                                ______________________________________                                        CNS white matter                                                                         211 ± 60 158 ± 45                                                                             242 ± 51                                  CNS gray matter                                                                           845 ± 106                                                                             362 ± 65                                                                             460 ± 55                                  Optic nerve culture                                                                      240 ± 67 272 ± 52                                                                             332 ± 58                                  Sciatic nerve culture                                                                    1,623 ± 173                                                                            1,850 ± 250                                                                          1,261 ± 141                               Trout CNS myelin                                                                         1,050 ± 110                                                                            1,150 ± 135                                                                          1,585 ± 185                               ______________________________________                                         *Protein (source) amounts were 100 μg (total protein liposomes) and 50     μg (gelapplied protein). Sample preparation was as described supra in      the Materials and Methods section. Tested protein, if not indicated           otherwise, was obtained from rat tissues.                                

Gray matter-derived liposomes contained markedly less nonpermissiveactivity. Significantly, high quantities of inhibitory activity wereextracted from optic nerve-derived cell cultures. Such cultures containhighly nonpermissive, myelin marker-positive oligodendrocytes (seeSection 6.2.1., supra). Analogous protein fractions from a Schwanncell-containing culture yielded no inhibitory proteins. Thus,nonpermissive substrate activity in the nervous system, as detected byour assay, codistributes with CNS white matter and with myelin-formingoligodendrocytes.

7.3. DISCUSSION

In this study, we have determined what makes rat CNS myelin a poorsubstrate. We first showed that brief treatment of the myelin withprotease abolished nonpermissiveness, demonstrating the involvement ofprotein. These proteins require detergent to be separated from themyelin membranes. Solubilized myelin protein reconstituted with aphosphatidylcholine/phosphatidylserine mixture yielded liposomes withhighly nonpermissive substrate properties. Liposomes with suchunfavorable substrate properties were obtained from rat CNS myelinprotein but not from the protein constituents of membrane fractionspossessing permissive substrate properties (PNS myelin, liver). We,therefore, assume that nonpermissiveness is due to the same protein(s)in myelin and in myelin-derived liposomes. When myelin proteins werefractionated by SDS-PAGE, protein fractions with relative molecularmasses of )35 kD and 250 kD were found to yield highly nonpermissiveliposomes upon reconstitution. Furthermore, nonpermissive kD and 250 kDprotein fractions could not be found in rat PNS myelin nor in aliver-derived membrane fraction. Therefore, the presence ofnonpermissive 35 kD and 250 kD proteins and nonpermissive membranefractions are correlated. Both protein fractions can functionindependently.

We have determined that the presence of the 35 kD and 250 kD proteinsfrom CNS myelin can be sufficient to generate a nonpermissive substrateby combining them with otherwise permissive substrate fractions. Thus,not only is nonpermissiveness of depleted rat CNS myelin restored (notshown), but supplemented liver or siatic nerve protein-derived liposomesbecome nonpermissive (FIG. 12). We therefore conclude that 35 kD and 250kD proteins of rat CNS myelin are likely to be responsible for itsnonpermissive substrate properties and that these proteins can beconsidered inhibitors of fibroblast spreading and of neurite outgrowth.Proof that the proteins are indeed the cause of CNS white matternonpermissiveness was obtained by use of specific blocking antibodies;such antibodies neutralized the nonpermissiveness of gel-purifiedinhibitors-containing liposomes, of CNS myelin membranes, and of livingcultured oligodendrocytes (as described in Section 8, infra).

8. ANTIBODY AGAINST MYELIN-ASSOCIATED INHIBITOR OF NEURITE GROWTHNEUTRALIZES NONPERMISSIVE SUBSTRATE PROPERTIES OF CNS WHITE MATTER

The examples described herein (Caroni and Schwab, March 1988, Neuron1:85-96) demonstrate that an inhibitory substrate mechanism preventsneurites from growing into optic nerve explants in vitro, over living,cultured oligodendrocytes, and over myelin used as a culture substrate.

CNS white matter from higher vertebrates and cultured differentiatedoligodendrocytes are nonpermissive substrates for neurite growth andfibroblast spreading. Monoclonal antibodies, termed IN-2 and IN-1, wereraised against 35 kD and 250 kD proteins respectively with highlynonpermissive substrate properties extracted from CNS myelin fractions.IN-1 and IN-2 bound both to the 35 kD and 250 kD inhibitors and to thesurface of differentiated cultured oligodendrocytes. Adsorption ofnonpermissive CNS myelin or nonpermissive oligodendrocytes with eitherantibody markedly improved their substrate properties. Optic nerveexplants injected with IN-1 or IN-2 allowed axon ingrowth of coculturedsensory and sympathetic neurons. We conclude that the nonpermissivesubstrate properties of CNS white matter are due to these membraneproteins on the surface of differentiated oligodendrocytes and to theirin vivo product, myelin.

8.1. EXPERIMENTAL PROCEDURES 8.1.1. CELL CULTURE

Mouse NIH 3T3 cells were cultured and assayed for spreading behavior inDMEM containing 10% FCS. SCGs from newborn rats were dissected anddissociated into single cells as described (Mains and Patterson, 1973,J. Cell. Biol. 59:329-345; Schwab and Thoenen, 1985, J. Neurosci.5:2415-2423). Neurons were cultured in an enriched L15 medium (Mains andPatterson, supra) with 5% rat serum and 100 ng/ml of 2.5S NGF.Overgrowth by non-neuronal cells was prevented by inclusion of cytosinearabinoside (10⁻⁵ M) in the culture medium. Inhibitoryoligodendrocyte-containing cultures were prepared from the optic nervesof 8-10 day old rats as described (see Section 6.1.1., supra). Opticnerve cultures were maintained in an enriched L15 medium with 5% ratserum. P3U myeloma cells and their hybridomas were cultivated in Iscovemedium supplemented with glutamine, antibiotics, 10⁻⁴ Mβ-mercaptoethanol, and 10% human serum.

8.1.2. SUBSTRATE PREPARATION

Myelin fractions were isolated as described in Section 6.1.5., supra).Briefly, carefully cleaned adult rat spinal cord tissue (CNS myelin) orrat sciatic nerve (PNS myelin) was homogenized in isotonic sucrosebuffer with a polytron homogenizer, and myelin membrane fractions wereobtained by flotation of low speed supernatants to densities below thatof 0.85 M sucrose (Colman, et al., 1982, J. Cell Biol. 95:598-608). Allisolation media contained trasylol (100 U/ml), 5 mM iodoacetamide, and 5mM EDTA to reduce protease digestion (this reagent mixture is designatedherein as protease inhibitors). Finally, myelin fractions were washedhypotonically in 30 mM Hepes (pH 7.4) (medium A) plus proteaseinhibitors and frozen in aliquots at -80° C. To prepareprotease-digested myelin membranes, the myelin was washed in medium Awithout protease inhibitors and subsequently incubated at aconcentration of 1 mg/ml in the presence of 0.1% trypsin (Sigma). After10 minutes at room temperature, 0.2% trypsin inhibitor (Sigma) was addedand membranes were washed free of protease in medium A. Oxidativechemical deglycosylation of myelin membranes was performed by theperiodate method as described (Beeley, 1985, in Laboratory Techniques inBiochemistry and Molecular Biology, R. H. Burdon and P. H. VanKnippenberger, eds., Elsevier, Amsterdam, pp. 279-288).

Liposomes containing myelin membrane proteins were prepared by thecholate dialysis method (see Section 7.1.4, supra). Briefly, solubilizedprotein was precipitated in a 10-fold excess volume of ice-cold acetone.Precipitated protein was collected by centrifugation after a 15 hourincubation at 4° C. and resuspended in 2.5% cholate in medium A. A 5- to50-fold excess (v/v) of phospholipids(phosphatidylcholine/phosphatidylserine, 10:1) in medium A plus 2.5%cholate was then added to the solubilized protein. The lipid-proteinmixture was applied to a Sephadex G50 column equilibrated in medium Aand liposomes were collected in the void volume. When membrane fractionswere used as protein source, precipitated protein was resuspended atapproximately 1 mg/ml, while gel-extracted protein was resuspended atapproximately 50 μg/ml.

Gel extraction of inhibitory proteins from CNS myelin was performed asdescribed (see Section 7.1.5, supra). Myelin protein was fractionated on3%-15% gradient gels under reducing conditions. For this purpose,samples were preincubated for 30 minutes at room temperature in SDS-PAGEsample buffer containing 2% SDS and β-mercaptoethanol. Protein from gelregions to be analyzed was extracted in the presence of 0.5% SDS and 50μg/ml insulin (Sigma). The latter was included to reduce losses due toadsorption of protein from low concentration solutions. The addedinsulin was removed from the protein to be tested by the Sephadex G50column (liposome formation procedure). When gel-extracted protein wasused as immunogen, no insulin was included in the gel extraction medium.

8.1.3. IMMUNOLOGICAL METHODS

The following antisera and monoclonal antibodies were used in thisstudy; anti-N-CAM (neural cell adhesion molecule) antiserum (gift of M.Schachner, Heidelberg, FRG), anti-tenascin antiserum (gift of R.Chiquet-Ehrismann, Basel, Switzerland), anti-J1 antiserum (gift of M.Schachner), monoclonal antibodies O₄ (anti-sulfatide) and O₁(anti-galactocerebroside)(gifts of M. Schachner).

Anti-CNS myelin antiserum was produced in rabbits by the injection of200 μg of myelin protein per immunization step. The cold-solublefraction of the antiserum was heat-inactivated by incubation at 56° C.for 1 hour.

To produce anti-inhibitory substrate monoclonal antibodies, BALB/c mice(6 week old females) were injected with approximatly 50 μg ofgel-extracted inhibitory (35 kD or 250 kD) fraction from rat CNS myelin.Gel-extracted protein was precipitated in acetone and resuspended at 1mg/ml in sterile PBS plus 0.1% cholate. Mice were immunized twice at 3week intervals, sera were tested for production ofnonpermissiveness-neutralizing antibodies (see also below), and micewith strong neutralizing sera were used for hybridoma production.

8.1.3.1. RADIOIMMUNOASSAY

Antibody presence was detected by a solid phase radioimmunoassay(Carlson and Kelly, 1983, J. Biol. Chem. 258:11082-11091) using ¹²⁵I-labeled goat anti-mouse antibody (Bio-Rad) as a probe.

Wells of 96 well plates were coated by exposing them to appropriateantigen (2 μg of protein per ml of 30 mM Tris [pH 7.4], 160 mM NaCl[Tris-saline]; 50 μl per well) for at least 3 hours at room temperature.Coated wells were then washed in Tris-saline plus 1% BSA, incubated inthe presence of the hybridoma supernatants, and finally assayed for thepresence of bound antibody with ¹²⁵ I-labeled goat anti-mouse antibody(approximately 10⁵ cpm per well). Values obtained with differentantigens cannot be compared quantitatively, as adsorption to the wellsvaried among different antigens. Background values for goat anti-mousebinding in the absence of mouse antibodies were routinely subtracted.Signals of less than 2 times background values (100-150 cpm) wereconsidered nonsignificant.

8.1.3.2. IMMUNOBLOTS

Transfer of CNS myelin protein fractionated by SDS-PAGE ontonitrocellulose was performed in 50 mM sodium phosphate buffer (pH 5.5),2 mM EDTA, 0.05% SDS (Filbin, M. T. and Poduslo, S. E., 1986, Neurochem.Int. 9:517-520). Transfer time was 3 hours at 1.6 A. Incubationprocedures with antibodies and ¹²⁵ I-labeled second antibody followedstandard procedures. Antibody-binding protein bands were visualized byautoradiography using high sensitivity Kodak X-ray films (X-OMAT).

Preparation of cell cultures for immunofluorescence microscopy wasperformed as follows. Cultures were rinsed in PBS and pre-fixed at 37°C. with fixation medium containing 4% paraformaldehyde. Upon extensiverinsing with PBS, cultures were incubated with the first antibody for 45minutes at room temperature (antibody dilutions, 1:50 for antisera andascites; 1:3 for hybridoma supernatants; dilution buffer consisted ofisotonic, sucrose-containing phosphate buffer (pH 7.4) plus 5% BSA).Unbound antibody was removed with 5% BSA-containing medium. After thusstaining the cells with monoclonal antibodies, the cells were incubatedwith a 1:50 dilution (in 5% BSA-containing medium) of the secondantibody, a rabbit anti-mouse antibody (SAKO, Copenhagen, Denmark) in 5%BSA-containing medium. The rabbit anti-mouse incubation enhanced signalintensities and was performed for 30 minutes at room temperature. Cellswere then fixed in isotonic buffer containing 4% paraformaldehyde.Fixation was interrupted after 30 minutes. Upon incubation withappropriate fluorescently labeled second antibody and subsequent washesin dilution buffer, bound antibody was visualized on an Olympus Vanoxfluorescence microscope. Control experiments with hybridoma medium wereperformed to exclude nonspecific fluorescent signals. Highly branchedoligodendrocytes or HBOs were identified by their characteristicmorphology and by double labeling experiments with the antibody O₁ (seeSection 6.1.3 supra).

Laminin was visualized by indirect immunofluorescence using a rabbitantiserum against EHS-tumor laminin on frozen sections of freshlydissected adult rat optic and sciatic nerves and on sections of nervesafter 4 weeks in culture (see below).

Immunoprecipitation of IN-1-binding (and of IN-2-binding) proteins wasperformed in immunoprecipitation buffer consisting of 150 mM NaCl, 30 mMHepes (pH 8.2), 2% NP40™, 0.5% sodium deoxycholate, plus proteaseinhibitors. Antibody was bound either to solubilized CNS myelin protein(solubilization in immunoprecipitation buffer for 1 hour at 4° C.) or tointact myelin membranes. In both cases, 100 μg of myelin protein wasincubated with 1 ml of hybridoma supernatant for 1 hour at roomtemperature. Myelin membranes incubated in the presence of antibody werewashed twice in medium A and solubilized in immunoprecipitation buffer.Rabbit anti-mouse was added in both 3.5 immunoprecipitation protocols tosolubilized antigen-antibody complex (20 μg of rabbit anti-mouse per mlof hybridoma supernatant), and incubation was allowed to proceed for anadditional 1 hour period at room temperature. Antigen-antibody-rabbitanti-mouse complex was finally sedimented with S. aureus cells (Sigma).Elution was performed by boiling for 5 minutes in 100 mM ammoniumchloride (pH 11.5) plus 0.5% SDS and β-mercaptoethanol. This procedureirreversibly inactivated present antibodies. Eluted, neutralized antigenwas precipitated with acetone and assayed for its substrate propertiesupon reconstitution into liposomes.

8.1.4. SUBSTRATE TESTING PROCEDURES

Substrates were tested as described in Section 7.1.2 supra). Myelinfractions or liposomes in medium A were dried onto the wells ofpolylysine-coated Greiner dishes (Greiner, Murtingen, FRG). Unboundmembranes were removed by 3 washes in Ca²⁺ - and Mg²⁺ -free Hank'smedium, and substrate-testing cells, i.e., 3T3 fibroblasts or superiorcervical ganglian SCG neurons, were immediately added to the dishes.When substrates were tested in the presence of antibody, boundsubstrates were incubated in the presence of undiluted hybridomasupernatants or in the presence of 1:30 dilutions (in Hank's medium) ofantisera. After 15 minutes at 37° C., four-fifths of theantibody-containing medium was removed and substituted withcell-containing medium. An analogous preincubation procedure was used inexperiments aimed at testing HBO nonpermissiveness. 3T3 experiments wereusually scored after a culture period of 1 hour, whereas SCG neuronswere allowed to grow processes for 24 hours in culture. When 3T3 cellswere preincubated with hybridoma supernatant, incubations were performedin gently agitated suspensions for a period of 15 minutes at roomtemperature. Cells were then sedimented, resuspended in culture medium,and added to substrate-adsorbed Greiner dish wells. 3T3 cells were addedat a density of 30,000 cells per cm², and SCG neurons were added at adensity of 20,000 cells per cm².

Quantitative evaluation of spreading was performed with a surfaceintegration program on at least 30 cells per experimental point.Photographs of randomly selected fields were used for this purpose, andthe outlines of all spread cells present in the field were tracedmanually with a graphic stylus connected to a computer. Only spreadcells were considered, and a zero-spreading value (strongly refractoryand round cells) was subtracted. Experiments were found to be subject toonly small quantitative variations, and values from representativeexperiments are given. Spreading degrees are given as means plus orminus standard error of the mean.

Quantitative evaluation of HBO inhibition of fibroblast spreading wasperformed by determining areas of overlap between O₁ ⁺ oligodendrocytesand 3T3 fibroblasts 1-2 hours after the addition of 3T3 cells to a 2 dayold optic nerve culture. The ratio of 3T3-oligodendrocyte overlap to thetotal area of oligodendrocytes was compared with the proportion of thetotal examined culture area occupied by 3T3 cells. Zero inhibition wasdefined as the absence of apparent discrimination by spreadingfibroblasts against surface occupied by oligodendrocytes.

8.1.5. NEURITE GROWTH INTO OPTIC NERVE EXPLANTS IN VITRO

Optic nerves of young adult rats were cultured together with dissociatedrat sympathetic or sensory neurons as previously described (Schwab andThoenen, 1985, J. Neurosci. 5:2415-2423). Briefly, optic nerves wererapidly dissected from 6-8 week old female rats, cleaned from adheringmeninges, and injected from both sides using a 10 μl Hamilton syringewith 2 μl of either IN-1 or IN-2 hybridoma supernatant or (controls)with O₁ hybridoma supernatant or antibody-free hybridoma medium. ATeflon ring with silicon grease separated the three chambers, and opticnerves were placed through the silicon grease, reaching from the middlechamber into one or the other side chamber. The respective antibodieswere present in the side chambers at a dilution of 1:10 throughout theculture period. Dissociated newborn rat SCG or dorsal root ganglionneurons were plated in the central chamber in L15 medium with rat serumand NGF, thus having access to one end of both nerves. After 3 weeks inculture, cultures were fixed with 2.5% glutaraldehyde and disassembled.The nerve explants were separately embedded in EPON. Sections forelectron microscopy were cut from three regions of each nerve; withinthe first 1 mm in the central chamber, from the region of the nerveunder the Teflon ring, and at a distance of 3 mm from the centralchamber end of the nerve (side chamber region). Most of the sectionscomprised the entire nerve in cross sections; they were sytematicallyscreened for the presence of axons using electron microscopy.

8.2. RESULTS 8.2.1. ANTISERUM AGAINST MYELIN NEUTRALIZES THENONPERMISSIVE SUBSTRATE EFFECTS OF CNS MYELIN AND AND OF HBOs

Antisera were generated against rat CNS myelin and adsorbed topolylysine-bound myelin. The antibody-adsorbed myelin was then assayedfor its substrate properties in supporting fibroblast spreading. Theantiserum contained antibodies that neutralized CNS myelinnonpermissiveness. Nonimmune rabbit serum did not significantly modifymyelin substrate properties. In these experiments, care was taken to,first, heat-inactivate rabbit serum fractions in order to prevent highlytoxic complement reaction and, second, to deplete the same fractions ofcold-insoluble proteins, which included fibronectin as a strong promoterof fibroblast spreading and attachment. The antiserum was also veryeffective in neutralizing HBO nonpermissiveness. Neutralization wasspecific, as rabbit antiserum against tenascin (Chiquet-Ehrismann etal., 1986, Cell 47:131-139) (FIGS. 14A-F), N-CAM (neural cell adhesionmolecule), and J1 (Kruse et al., 1985, Nature, 316:146-148) did notinfluence myelin or HBO substrate properties. The cell adhesionmolecules N-CAM and J1 were present in substantial amounts on thesurface of HBOs as detected by immunofluorescence. Tenascin antigenicitywas absent from the myelin (shown by radioimmunoassay) as well as fromthe HBO surface (shown by immunofluorescence). This finding is importantas it demonstrates that the documented, unfavorable substrate propertiesof tenascin (Chiquet-Ehrismann et al., 1986, Cell 47:131-139) are notresponsible for myelin or HBO substrate properties. The experiments withmyelin-antiserum demonstrated that the properties of both testedsubstrates, myelin and oligodendrocytes, could be improved by antibodybinding.

8.2.2. IN-1: A MONOCLONAL ANTIBODY AGAINST GEL-PURIFIED 250 kD INHIBITORFRACTION FROM CNS MYELIN NEUTRALIZES MYELIN NONPERMISSIVENESS

Mice were immunized with rat CNS myelin 250 kD protein fraction,hybridomas were raised, and supernatants were screened for anti-myelinantibodies. Positives were rescreened for neutralization of CNS myelinnonpermissiveness by the 3T3 cell spreading assay. Five myelin-positiveantibodies fulfilled the second screening criterion to varying degrees.The antibody with the strongest neutralizing properties was selected anddesignated IN-1.

Adsorption of liposomes containing the 250 kD protein, liposomescontaining the 35 kD protein, and rat CNS myelin with IN-1 drasticallyreduced nonpermissiveness in all three cases (FIGS. 13A-H).Neutralization was slightly less efficent for CNS myelin membranes(FIGS. 13A and 13B), possibly due to incomplete saturation of inhibitorysites by the antibody. The antibody bound to inhibitor-containingliposomes and to CNS myelin, but not to PNS myelin (Table V).

                  TABLE V                                                         ______________________________________                                        IN-1 BINDS TO 35 kD AND 250 kD                                                MEMBRANE PROTEINS FROM RAT CNS MYELIN*                                                    (Amount of Antibody Bound                                                     cpm of .sup.125 I-Labeled Goat Anti-Mouse)                        Antigen       IN-1      1G9**     O1**                                        ______________________________________                                        CNS myelin:                                                                   Control       550 ± 35                                                                             1500 ± 110                                                                           10850 ± 550                              Trypsin-treated                                                                              25 ± 20                                                                             40 ± 30                                                                              8200 ± 480                               PNS myelin:    80 ± 25                                                                             350 ± 30                                                                             9200 ± 500                               Liposomes containing:                                                         250 kD CNS myelin protein                                                                   250 ± 30                                                                             45 ± 20                                                                               80 ± 50                                 35 kD CNS myelin protein                                                                    350 ± 35                                                                             40 ± 30                                                                               90 ± 60                                 ______________________________________                                         *Antibody binding sites were detected by a solid phase radioimmunoassay       using .sup.125 Ilabeled goat antimouse antibody as a probe. Liposomes wer     prepared from gelextracted CNS myelin protein (100 μg of myelin protei     added to the gel). Values are given after subtraction of background           binding in the absence of primary antibody. Background values for             liposomes were essentially identical to the ones obtained with antibody i     the presence of proteinfree liposomes,  #  i.e., approximately 120-150        cpm.                                                                          **Antibody 1G9 is an antimyelin monoclonal antibody that binds to the         surface of differentiated oligodendrocytes and to protein of 110 kD on        Western blots of rat CNS myelin.                                         

Neutralization of inhibitory substrate properties of myelin fractionswas observed for superior cervical ganglion (SCG) neurons (FIGS. 13A-h),for 3T3 fibroblasts (see Table VI), and for neuroblastoma cells in thepresence of dibutyryl-cAMP.

                  TABLE VI                                                        ______________________________________                                        IN-1 NEUTRALIZES NONPERMISSIVENESS OF CNS MYELIN                              AND ITS SPREADING INHIBITORS OF 35 kD AND 250 kD*                                       3T3 Spreading (μm.sup.2)                                         Substrate   Control     +IN-1     O1                                          ______________________________________                                        CNS myelin  278 ± 31 1446 ± 114                                                                           250 ± 36                                 250 kD liposomes                                                                          213 ± 11 1335 ± 151                                                                           245 ± 35                                 35 kD liposomes                                                                           185 ± 18 1286 ± 113                                                                           210 ± 21                                 Protein-free liposomes                                                                    1520 ± 145                                                                             1410 ± 105                                                                           1495 ± 145                               ______________________________________                                         *Spreading extents were estimated after 1 hour in culture in the presence     or the absence of hybridoma supernatant. Substrate protein amounts            adsorbed to the wells of Greiner dishes were as follows:                      CNS myelin, 20 μg per cm.sup.2 ;                                           liposomes, 100 μg of CNS myelin protein were applied to the gel lane       from which proteins of indicated apparent molecular weight were extracted     and the entire extracted and reconstituted protein was applied to the         culture well. Apparent molecular weight ranges were estimated with            molecular weight standards (BioRad) and were about 35 ± 3 kD and 250       ± 15 kD, respectively.                                                

protein-free lipid vesicle, or on permissive liposomes containingperipheral 250 kD protein (see FIGS. 13A-H). In these latter cases, cellattachment and spreading were slightly impaired, but no improvement wasobtained with IN-1, demonstrating the specificity of the antibody effectfor neutralizing myelin-derived inhibitors. Neutralization was due tothe antibody fraction in IN-1-containing supernatants, as an ammoniumsulfate-precipitated fraction of IN-1 ascitic fluid was equallyeffective.

IN-1 binding is completely abolished by a brief pretreatment of themyelin with trypsin, demonstrating that the antibody does not bind toglycolipids (Table V). IN-1 antibody when preadsorbed ontoHBO-containing cultures also efficiently reduced HBO nonpermissiveness(see FIGS. 14A-F). In control experiments, 3T3 cells never invaded morethan 10% of the surface of O₁ ⁺ (galactocerebroside; marker fordifferentiated oligodendrocytes) HBOs. Fibroblasts seeded directly ontoHBOs failed to spread and eventually detached. Often more than 50% ofHBO surface was covered by fibroblasts in the presence of IN-1. Inaddition, spreading of fibroblasts on antibody-adsorbed HBOs wasfrequently observed (FIGS. 14A-F). Interestingly, quantitativedetermination of the substrate properties of HBOs in the presence ofIN-1 showed that 3T3 cells prefer HBOs over the polylysine-coatedculture dish under this condition (FIGS. 14A-F). This behavior could berelated to the presence of cell adhesion molecules like myelinassociated glycoprotein, J1, or N-CAM on these oligodendrocytes. FIG. 16also shows that IN-1 bound to the surface of living HBOs. Specificstaining of intact cells with the morphology of astrocytes, fibroblasts,or immature, A₂ B₅ ⁺ oligodendrocytes was not detected by our method.Also, no specific IN-1 staining could be detected on the surface ofliving neuronal cells or neuroblastoma cells. The observed weak stainingof HBOs was probably due to the fact that spreading inhibitors are minorproteins in myelin and optic nerve culture fractions. These experimentsdemonstrate that nonpermissiveness in HBOs is, as previously shown formyelin, an IN-1-affected process.

8.2.3. 250 kD AND 35 kD INHIBITORS FROM CNS MYELIN SHARE TWONEUTRALIZING EPITOPES

As shown in Table VI, IN-1 did bind to liposomes containing the 35 kDinhibitor. Therefore, the epitope defined by IN-1 is shared between the250 kD and 35 kD inhibitors (see also FIG. 16). Such an epitope may be apolypeptide since treatment of the myelin with periodate to removecarbohydrate did not affect IN-1 binding. Table VI demonstrates that the35 kD inhibitor was neutralized by IN-1. As the antibody neutralized theinhibitory substrate properties of myelin membranes, these experimentsare consistent with the interpretation that both the 250 kD and the 35kD inhibitor contribute to myelin nonpermissiveness.

Control experiments (Table VI) excluded that IN-1 neutralization was dueto nonspecific masking of the myelin as a consequence of antibodybinding, as monoclonal antibodies O₁ and O₄, which bind to very abundantantigens on the surface of myelin and HBOs, did not reduce thenonpermissive substrate effects of either myelin (Table VI, for O₁) orliving HBOs (FIG. 15, for O₄).

Immunization experiments, as previously described for the 250 kDprotein, were performed with the gel-purified kD inhibitor fraction fromrat CNS myelin, confirming the relatedness of the 250 kD and 35 kDmyelin inhibitors. Hybridomas produced from such mice were tested asdescribed for monoclonal antibody IN-1. The strong neutralizing antibodyIN-2 was selected. Neutralization and binding properties of IN-2 aresummarized in Table VII.

                  TABLE VII                                                       ______________________________________                                        IN-2 BINDS TO CNS MYELIN INHIBITORY                                           SUBSTRATES AND NEUTRALIZES THEIR                                              NONPERMISSIVE SUBSTRATE PROPERTIES*                                                     IN-2 Binding                                                                  (.sup.125 I-Labeled Goat                                                                 3T3 Spreading (μm.sup.2)                              Antigen/Substrate                                                                         Anti-Mouse cpm)                                                                            No IN-2   +IN-2                                      ______________________________________                                        CNS myelin  975          245 ± 15                                                                             1300 ± 121                              CNS myelin, 100          1485 ± 110                                                                           1430 ± 158                              trypsin-treated                                                               CNS myelin liposomes                                                                      700          180 ± 18                                                                             1040 ± 110                              35 kD liposomes                                                                           285          215 ± 15                                                                             1250 ± 120                              250 kD liposomes                                                                          410          205 ± 11                                                                             1180 ± 108                              Protein-free liposomes                                                                    100          1385 ± 125                                                                           1420 ± 160                              ______________________________________                                         *Experimental details are described supra in Experimental Procedures and      Tables V and VI. Data presented in the table were obtained with a 1:100       dilution (in PBS) of ammonium sulfateprecipitated antibody from ascitic       fluid (10 μg of protein per ml after dilution). CNS myelin liposomes       were formed from 20 μg of CNS myelin protein                          

Thus, IN-2 bound to both the 35 kD and the 250 kD protein, itneutralized nonpermissiveness of myelin membranes and HBOs, and it boundto the surface of living HBOs. IN-1 and IN-2 epitopes are not identical:IN-2, but not IN-1, strongly bound to cytoskeleton-associated antigenswhen astrocytes or fibroblasts were permeabilized. As found for IN-1,IN-2 did not bind to protease-treated myelin.

8.2.4. IN-1 SPECIFICALLY IMMUNOPRECIPITATES NONPERMISSIVE SUBSTRATEACTIVITY FROM SOLUBILIZED MYELIN PROTEIN

Since the inhibitory protein fractions used in this study apparentlycontained more than one protein species, immunoprecipitation experimentswere performed to determine whether IN-1 binds directly to neuritegrowth- and fibroblast spreading-preventing protein(s).

Solubilized myelin protein was adsorbed with IN-1 antibody, andantigen-antibody complex was sedimented with rabbit anti-mouse bound toStaphylococcus aureus cells. Antigen-antibody complexes were thendissociated under denaturing and reducing conditions to irreversiblyinactivate the IN-1 antibody. Liposomes formed in the presence ofimmunoprecipitated protein were highly inhibitory for fibroblastspreading (Table VIII).

                  TABLE VIII                                                      ______________________________________                                        IN-1 SPECIFICALLY REMOVES INHIBITORY SUBSTRATES                               FROM CNS MYELIN PROTEIN, INHIBITORY PROTEINS                                  OF 35 kd AND 250 kd ARE IMMUNOPRECIPITATED BY IN-1*                                          3T3 Spreading (μm.sup.2)                                    Myelin Protein Fraction                                                                        Protocol 1                                                                              Protocol 2                                         ______________________________________                                        Total            322 ± 32                                                                             255 ± 25                                        IN-1-depleted    1361 ± 61                                                                            1280 ± 90                                       O.sub.1 -depleted                                                                              612 ± 63                                                                             ND                                                 IN-1-immunoprecipitated                                                                        218 ± 16                                                                             215 ± 18                                        O.sub.1 -immunoprecipitated                                                                    1218 ± 108                                                                           1410 ± 128                                      From IN-1-precipitated:                                                       250 kD liposomes ND        245 ± 18                                        35 kD liposomes  ND        270 ± 21                                        ______________________________________                                         *Details of immunoprecipitation protocols are given in Section 8.1.3.2.,      supra.                                                                        Protocol 1: immunoprecipitation from solubilized CNS myelin protein.          Protocol 2: immunoprecipitation upon binding of antibody to intact myelin     membranes and subsequent solubilization of antigenantibody complexes. In      both protocols, immunoprecipitation was performed using 100 μg of CNS      myelin protein. Total refers to liposomes formed from 1% of the starting      material.  #  Liposome fractions designated depleted were formed from 1%      of the immunoprecipitation supernatant. Those designated                      immunoprecipitated were formed from 5% of the immunoprecipitated and          eluted material. Finally, in the experiments presented in the second part     of the table, IN1-immunoprecipitated protein (from 500 μg of starting      CNS myelin protein) was separated by SDSPAGE, and gel regions of indicate     apparent molecular weight were extracted from  #  the gel and                 reconstituted as described in Section 8.1.3.2., and previous tables. In       these cases, liposomes supra corresponding to 5% of the gelextracted          protein were adsorbed to the 1 cm.sup.2 wells.                                ND, not determined.                                                      

In control experiments, both O₁ antibody (Table VIII) and a monoclonalantibody against 110 kD myelin protein did not immunoprecipitateinhibitory substrate.

Immunoprecipitation of inhibitory substrate with IN-1 could also beperformed when antibody was first bound to myelin membranes (TableVIII). In the latter case, membranes were subsequently washed free ofunbound antibody, myelin protein was solubilized, and antigen-antibodycomplex was sedimented as described above. This experiment demonstratedthat the inhibitory substrate-associated IN-1 epitope(s) was accessibleto antibody in its native myelin membrane location. Whenimmunoprecipitated proteins were subsequently separated by SDS-PAGE,inhibitor-containing protein fractions of 35 kD and of 250 kD could beextracted from the gel (Table VIII). Therefore, 35 kD and 250 kDinhibitors of neurite growth and fibroblast spreading expose the IN-1epitope on the surface of myelin membranes.

FIG. 16 shows that IN-1 binding to CNS myelin proteins fractionated bySDS-PAGE and adsorbed onto nitrocellulose was restricted to a subset ofminor myelin proteins. While binding to 250 kD protein was consistentlyobserved, binding to protein in the 35 kD region was weak and often notdetectable. In addition, IN-1 bound to 56 kD protein, which had notpreviously been recognized as yielding inhibitory liposomes uponreconstitution. When myelin fractions yielding strong IN-1⁺ 56 kDprotein were used as the source of gel-purified 56 kD protein, highlyinhibitory liposomes were obtained upon reconstitution. Whileessentially no binding of IN-1 to intact, permissive PNS myelinmembranes could be observed (Table V), Western blot analysis of PNSmyelin protein with IN-1 revealed immunoreactive material of 300-400 kD.Upon gel extraction and reconstitution, this latter material yieldedliposomes with permissive substrate properties. Masking of ahypothetical inhibitory substrate by highly favorable substrate from thesame 300-400 kD PNS myelin protein region cannot be excluded. However,attempts to immunoprecipiate inhibitory substrate from solubilized PNSmyelin protein with IN-1 have failed. It therefore seems reasonable toassume that the antibody IN-1 also binds to proteins with no inhibitorysubstrate properties. Therefore, identification of inhibitory substratesby Western blot analysis with the antibody IN-1 is presently notwarranted.

8.2.5. NONPERMISSIVENESS OF ADULT OPTIC NERVE IS NEUTRALIZED BYADSORPTION WITH IN-1 ANTIBODY

Optic nerve explants, in contrast to sciatic nerve explants, have beenobserved not to support growth of neurites in vitro, even when optimalamounts of appropriate neurotrophic factor was present (Schwab andThoenen, 1985, J. Neurosci. 5:2415-2423). Cultured optic nerve explantswere assayed for laminin immunoreactivity as neurite growth is known tobe supported by laminin and furthermore since laminin is known to bepresent in sciatic nerve but not in optic nerve in situ. Laminin wasexclusively present on the pial basement membrane and around bloodvessels when freshly dissected optic nerve from adult rat was analyzed.The explant, however, contained substantial amounts of stronglylaminin-positive cells, presumably astrocytes, after 3-4 weeks in vitro(FIGS. 17A-B). Despite the presence of laminin, no neurites were foundto grow into optic nerve after periods of up to 5 weeks in vitro. Thesefindings supported the interpretation that a nonpermissive substratepresent in the optic nerve explants is responsible for its unfavorablemicroenvironment.

In subsequent experiments, IN-1 antibody was injected into the opticnerve explant prior to insertion of the explant in a three-compartmentchamber culture system (Schwab and Thoenen, 1985, J. Neurosci.5:2415-2423). In addition, IN-1-containing supernatant was also added tothe compartment containing the distal end of the nerves for the durationof the experiment. In control experiments, supernatants rich in O₁antibody were injected and included in the culture medium.

The results from these experiments demonstrated that IN-1, but not O₁antibody, effectively promoted extensive growth of sympathetic andsensory neurites into the optic nerves (Table IX, FIGS. 18A-B).

                  TABLE IX                                                        ______________________________________                                        INJECTION OF OPTIC NERVE EXPLANTS WITH                                        ANTIBODY IN-1 RESULTS IN INGROWTH OF                                          AXONS INTO OPTIC NERVE IN VITRO*                                                       Antibody IN-1    Antibody O.sub.1                                             Optic Region     Optic Region                                        Culture    1 mm   3 mm        1 mm 3 mm                                       ______________________________________                                        1          +++    +++         ++   -                                          2          ++     +++         +    +                                          3          ++     +++         +    +                                          4          ++     ++          +    -                                          5          +++    +++                                                         6          ++     ++          -    -                                          ______________________________________                                         *Optic nerve explants were injected with antibody IN1 or antibody O.sub.1     and then placed into chamber cultures with sensory neurons in the central     chamber. After 3 weeks in culture, nerves were systematically examined by     electron microscopy. Presence of axons at 1 and 3 mm in the optic nerves      of representative experiment.                                                 + indicates 1-20 axons;                                                       ++: 20-50 axons;                                                              +++: >50 axons per cross section.                                             Large numbers of axons were found deep in the IN1-injected nerves, but no     in the O.sub.1injected nerves.                                           

Neurites extended for lengths of more than 3 mm into optic nerves in thepresence of IN-1 (Table IX). Although preferred as a substrate, growthwas not restricted to regions adjacent to basal membrane, and contact ofingrowing neurites with myelin could frequently be observed (FIGS.18A-B). In some experiments, damaged control nerves with largetissue-free spaces did allow limited neurite growth. In those cases,however, neurites were not found in contact with myelin sheets. Neuritegrowth over a distance of 3 mm into the optic nerves was observed in 5out of 6 cases when IN-1 was present. Growth in O₁ -containing nerve wasobserved in 1 out of 5 cases (Table IX).

These findings strongly suggest that nonpermissiveness of optic nerveexplants in vitro is due to IN-1-binding inhibitory substrate. As 35 kDand 250 kD inhibitory substrates from CNS myelin are found in opticnerve tissue, these proteins are likely to be responsible for itsnonpermissive microenvironment in vitro and possibly also in vivo.

8.3. DISCUSSION

The experiments described herein demonstrate that monoclonal antibodiesraised against each gel-purified inhibitor fraction neutralized orgreatly reduced the nonpermissiveness of both inhibitors, of isolatedmyelin membrane fractions, of cultured HBOs, and of adult rat opticnerve explants. The antibodies bind to the surfaces of myelin membranesand cultured oligodendrocytes. They specifically immunoprecipitateinhibitory substrate proteins of 35 kD and 250 kD from myelin proteinfractions. We conclude that nonpermissiveness of adult CNS whitematter-derived tissues, cells, and subcellular fractions is due to thesame inhibitory substrate mechanism involving IN-1-binding (andIN-2-binding) proteins. Clearly, 35 kD and 250 kD inhibitors share twoantigenic sites, IN-1 and IN-2. In both cases, antibody bindingabolished nonpermissive substrate properties. Our data are consistentwith the interpretation that the proteins reponsible for adult CNS whitematter nonpermissiveness are the 35 kD and 250 kD (and 56 kD) inhibitorysubstrates extracted from rat CNS myelin.

9. INVOLVEMENT OF A METALLOPROTEASE IN GLIOBLASTOMA INFILTRATION INTOCENTRAL NERVOUS SYSTEM TISSUE IN VITRO

In the examples detailed herein, we describe a membrane-associatedmetalloprotease which plays a crucial role in the malignant tumorinfiltration of CNS tissue in vitro by the rat glioblastoma cell lineC6.

We have discovered that malignant tumor infiltration of CNS tissue invitro by the glioblastoma line C6, requires a plasma membrane boundmetallodependent degradative activity. C6 cells infiltrate optic nerveexplants, attach and spread on white and grey matter of cerebellarfrozen sections or on CNS myelin. The metal ions chelator1,10-phenanthroline and the dipeptide cbz-tyr-tyr, but not inhibitorsfor three other classes of proteases, blocked up to 67% of C6 cellspreading on CNS myelin. A 0metallodependent activity neutralizing CNSmyelin inhibitory substrate properties toward 3T3 cells, is associatedwith a C6 plasma membrane fraction. The same inhibitors ofmetalloprotease also impaired infiltration of CNS nerve explants andspreading on the CNS white matter of cerebellar frozen sections.

9.1. MATERIALS AND METHODS 9.1.1. CELL CULTURES

Rat C6, mouse NIH 3T3 and B16 cells were cultured in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS),usually to maximally 70-80% confluency. Cells were harvested with ashort trypsin treatment (0.1% in Ca²⁺ /Mg²⁺ -free Hank's medium for 90seconds) stopped by addition of FCS in excess, collected bycentrifugation. Cells were resuspended in either DMEM/FCS or definedserum-free medium (MEM) and used for experiments. Dissociated rat CNSglial cells were prepared starting from optic nerves of 6-7 days oldLewis rats as described in Section 6.1.1, supra and plated intopoly-D-lysine (PLYS) coated wells (100 mm², 100 μl medium) at a densityof 20,000 cells per well. The culture medium was an enriched L15 mediumwith 5% rat serum, penicillin and streptomycin. C6, 3T3 and B16 cellswere added to 2 day old cultures at a concentration of 30,000 cells perwell, incubated for two hours and fixed with warm 4% formalin inphosphate buffer. Inhibitory oligodendrocytes were identified by doublelabelling using the specific antibodies O₁ and O₄ (see Section 6.1.3,supra).

9.1.2. PREPARATION OF NERVE EXPLANTS FOR INFILTRATION ASSAY

Optic nerve and sciatic nerve explants were prepared as described(Schwab and Thoenen, 1985, J. Neurosci. 5:2415-2423). Briefly, thenerves were rapidly dissected from about 8 week old male rats, cleanedfrom the meninges, 3 times frozen and thawed using liquid nitrogen, andplaced under a teflon ring (diameter 13 mm, thickness 1 mm) sealed to aculture dish with silicon grease. Two chambers connected only by theexplants were in this way obtained. 300,000 C6, 3T3 or B16 cells wereplated in the inner chamber in DMEM/FCS and incubated for 5 to 20 days.The medium was changed every other day. Cultures were fixed overnightwith 4% formalin. The nerve explants were mounted with Tissue-Tek, 10 to15 μm sections were cut in a cryostate and collected on gelatine coatedcover slips. After drying at room temperature overnight, the sectionswere stained in 0.75% cresyl violet, and evaluated. The infiltratedcells were counted for each 0.1 mm of the explants, starting from thetip where cells were added. Due to the 15 day incubation, the explantswere often different in diameter. Therefore, only the central part ofthe nerves (0.25 mm) were considered, since only this part of theexplants presented a good histological quality. Inhibition experimentswere performed with nerve explants previously injected from both sideswith 2 μl of 3 mM cbz-tyr-tyr or cbz-ala-phe solutions.

9.1.3. CNS FROZEN SECTIONS AND MYELIN AS SUBSTRATES

Adult rat cerebellum frozen sections were prepared and dried on glasscoverslips. 70,000 C6, 3T3, or B16 cells in 100 μl were added to eachwell containing slices previously rinsed with cold DMEM/FCS. Cultureswere incubated for 2 days at 37° C. Cultures were then fixed and stainedwith cresyl violet. Three to four cerebellum slices were used per pointper experiment, with each experiment being repeated at least 2 times.

Myelin from rat spinal cord (CNS) or sciatic nerve (PNS) purified on adiscontinuous sucrose gradient as described in Section 6.1.5., was driedovernight onto PLYS coated wells (20 μg protein/well of 100 mm²surface). Unbound membranes were removed by three washes with Ca²⁺ /Mg²⁺-free Hank's solution. Myelin coated wells were immediately used insubstrate testing assays by the addition of 9,000 cells (C6, 3T3, orB16) per cm². Alternatively, we used extracted CNS myelin protein, orSDS-PAGE purified 35 and 250 kD inhibitory proteins reconstituted inliposomes (see Section 7.1.5, supra). Experiments were scored atdifferent time points using a phase contrast microscope equipped with aphotocamera. Quantifications were done using a surface integrationprogram; three arbitrary fields were photographed for each well at amagnitude of 80×, at least 25 cells per picture were measured. Eachpoint represents the mean of at least 3 wells±SEM. Results are expressedas μ² of projected cell surface, or as degree, which was calculated bysubtracting from the projected surface value of a spreading cell, thesurface value of a completely spheric cell.

9.1.4. C6 PLASMA MEMBRANES AND CONDITIONED MEDIUM PREPARATION

C6 cells grown to 80% confluency were washed twice with Hank's medium,and harvested in 20 ml 8.5% sucrose, 50 mM NaCl, 10 mM Tris buffer, pH7.4, using a rubber policeman. After mechanical homogenization through aseries of needles of decreasing size, a low purity plasma membranefraction was obtained by centrifugation (5 minutes at 3000× g, 10minutes at 8000× g, and then 2 hours at 100,000× g). A higher purityfraction was isolated by loading the material on a discontinuous sucrosegradient, containing 50 mM NaCl, 10 mM Tris, pH 7.4 (Quigley, 1976, J.Cell Biol. 71: 472-486). 20-40% sucrose interphase (C6 plasma membranesfraction) and 40-60% sucrose interphase (C6 mitochondrial fraction) werecollected, washed in Hank's medium and resuspended in MEM.

Conditioned media were obtained by cultivating 80% confluent C6 cellcultures for 1 day in MEM. The medium was then collected and centrifugedfor 10 minutes at 3000× g. In some experiments the conditioned mediumwas concentrated 10 times using Centricon Tubes.

9.1.5. TREATMENT OF CNS MYELIN WITH C6 PLASMA MEMBRANES

CNS myelin coated PLYS wells were prepared as described in the previoussection, but instead of being immediately tested as substrate, they werefirst incubated with 50 μl of C6 plasma membranes (containing 0.8 mgprotein/ml MEM) at 37° for 30 minutes. Dishes were then rinsed twicewith Hank's medium and immediately used as substrates for 3T3 cells. Insome experiments, protease blockers were added to the membranes using 10times concentrated solutions.

9.2. RESULTS 9.2.1. C6 GLIOBASTOMAS BUT NOT 3T3 FIBROBLASTS OR B16MELANOMAS INFILTRATE OPTIC NERVE AND CNS WHITE MATTER IN VITRO

Frozen optic nerve and sciatic nerve explants were placed under a teflonring and sealed with silicon grease (Schwab and Thoenen, 1985, J.Neurosci. 5:2415-2423). C6 or 3T3 cells were plated into the ring, incontact with one end of the nerve explants. Culture medium was exchangedevery other day, and after 5 to 20 days of incubation the nerves werefixed, and sectioned with a cryotome. Infiltrated cells were recognizedby cresyl violet staining. PNS explants supported diffuse infiltrationof both C6 and 3T3 cells (FIGS. 19C, D). C6 cells were present in theexplants at higher density. In the optic nerve explants, a differentpicture emerged (FIGS. 19A, B); 3T3 cells did not infiltrate the nerves,with the exception of very few cells which migrated along blood vessels(FIG. 19B, arrow). On the other hand, C6 cells infiltrated deep into theoptic nerves with a diffuse pattern, reaching a maximum distance ofabout 3 mm from the entry point in 14 days (migration rate: about 0.2mm/day).

As an alternative model, adult rat cerebellum frozen sections were usedas a culture substrate for C6, B16 or 3T3 cells. The highly metastaticB16 melanoma cells were found to clearly discriminate between thesubstrate qualities of the grey and white matter with regard to cellattachment, spreading and migration. In fact, B16 cells exclusivelyattached and spread on grey matter regions and, even if plated at highcell densities, they did not attach on or migrate into white matterareas of the sections (FIG. 20E). The same picture emerged for 3T3cells, which formed dense monolayers on grey matter, but not on whitematter (FIGS. 20C, D). In contrast to B16 and 3T3 cells, C6 cells werefound frequently on white matter as well as on grey matter (FIGS. 20A,B). In some cases we found that C6 cells were more dense on the whitematter than on the molecular layer of the grey matter, where they oftenformed little aggregates which spread with difficulty.

9.2.2. GLIOBLASTOMA CELL SPREADING IS NOT INHIBITED BY CNS MYELIN

The spreading behavior of C6 glioblastomas on CNS myelin adsorbed toPLYS coated wells was compared to that of B16 melanomas and 3T3fibroblasts. B16 melanoma reaction to a CNS myelin substrate stronglyresembled that of 3T3 fibroblasts: 3T3 or B16 cells spreading on CNSmyelin was strongly impaired, whereas C6 cell spreading was slightlyreduced at the beginning (90 minutes), but no further appreciabledifferences were detected at later time points (FIGS. 21A-C). Thedifferences between cells on CNS myelin or on PLYS also persisted withprolonged incubation times (up to 1 day).

C6 cells were confronted with the SDS-PAGE purified inhibitors (35 kDand 250 kD) reconstituted in liposomes, and also with living, culturedoligodendrocytes. Again, 35 kD and 250 kD liposomes strongly inhibited3T3 cell spreading, but they did not impair C6 cell spreading; C6 cellsadhered and rapidly assumed the well spread characteristic "fried egg"appearance also on these reconstituted CNS myelin fractions.

9.2.3. SPECIFIC BLOCKERS OF METALLOPROTEASES INHIBIT C6 CELL SPREADINGON CNS MYELIN

The involvement of proteases in C6 behavior was investigated bydetermining the effect of inhibitors of proteases on C6 cell spreadingon either CNS myelin or PLYS. Cys-, Ser- and Asp-protease blockers atthe adequate concentrations had no discernible effect on C6 spreading onCNS myelin (Table X).

                  TABLE X                                                         ______________________________________                                        EFFECT OF DIFFERENT PROTEASE INHIBITORS ON C6                                 CELL SPREADING ON PLYS OR CNS MYELIN*                                                            Spreading on:                                                                 PLYS  CNS    Inhibition                                    Protease                                                                            Protease           (% of control                                                                            on CNS                                    Class Inhibitor          on PLYS)   (%)                                       ______________________________________                                        none,                          100   95   5                                   control                                                                       serine                                                                              6-amino-capronate                                                                          3.0    mM   93    100  0                                         hirudine     1.0    nM   nq    nq   0                                         PMSF         4.0    mM   100   94   6                                         trasylol     200.0  U/ml 98    93   5                                   cysteine                                                                            leupeptine   0 3    mM   91    83   8                                   aspartic                                                                            pepstatine   0.3    mM   98    95   3                                   metallo                                                                             1,10-phenanthroline                                                                        0.3    mM   97    30   67                                        bestatine    0.1    mM   nq    104  0                                         phosphoramidon                                                                             0.3    mM   nq    91   9                                         TIMP         10.0   μg/ml                                                                           102   93   9                                         cm-phe-leu   0.5    mM   95    92   3                                         cbz-gly-gly-NH.sub.2                                                                       1.0    mM   nq    99   1                                         cbz-gly-phe-NH.sub.2                                                                       1.0    mM   100   45   55                                        cbz-ala-phe  0.3    mM   98    90   8                                         cbz-tyr-tyr  0.3    mM   101   56   45                                  general                                                                             2-macroglobulin                                                                            3.0    μM                                                                              70    52   18                                        cocktail -               nq    nq   0                                         cocktail +               nq    nq   ++                                  ______________________________________                                         *Cells were plated on PLYS or CNS myelin coated culture dishes.               Spreading was determined after 150 minutes as described supra in Material     and Methods. Inhibition values were calculated by subtracting spreading       values on CNS myelin from the values on PLYS.                                 PMSF: Phenyl methyl sulfonyl fluoride.                                        TIMP: Tissue inhibitor of metalloproteases.                                   Cocktail -: trasylol, 200 U/ml; leuptine, 0.3 mM; pepstatine, 0.3 mM.         Cocktail +: same as cocktail , but with 0.3 mM 1,10phenantroline.             nq: not quantified, only qualitative                                     

The specific metalloprotease blocker 1,10-phenanthroline on the otherhand, resulted in a strong inhibition of C6 spreading specifically onCNS myelin: 1,10-phenanthroline inhibited C6 spreading on myelin up to67% after 2 hours in culture (Table X). None of the blockers testedshowed a significant effect on C6 cell spreading on PLYS.1,10-phenanthroline is a general metalloprotease inhibitor due to itsproperty of metal ion chelation. However, inhibition by this substanceis not sufficient to define a proteolytic activity, since othermetallodependent enzymes are also inhibited. Many other inhibitors ofmetalloproteases have been found, but they usually turned out not to beas general as 1,10-phenanthroline. Phosphoramidon (Komiyama, et al.,1975, Biochem. Biophys. Res. Comm. 65:352-357), bestatine (Umezawa, etal., 1976, J. Antibiot. 29:857-859) and the tissue inhibitor ofmetalloprotease (TIMP; Cawston, et al., 1987, Biochem. J. 195:159-165)did not impair C6 cell spreading (Table X).

TIMP also does not inhibit a brain membrane associated metalloproteasedegrading enkephaline. Carboxymethyl-phe-leu (Fournie-Zaluski, M. C. etal., 1983, J. Med. Chem. 26:60-65), a modified peptide with highaffinity for enkephalinase (Almenoff, J. and M. Orlowski, 1983,Biochemistry 22:590-599), did not inhibit C6 cell spreading (Table X).On the other hand, we found that the dipeptides cbz-gly-phe-NH₂ andcbz-tyr-tyr lead to 55% inhibition of C6 cell spreading on CNS myelin,but not on PLYS, PNS myelin or glass. These peptides are substratepeptides with metalloprotease specificity (Almenoff and Orlowski, supra;Baxter, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4174-4178; Couchand Strittmatter, 1983, Cell 32:257-265; Chen and Chen, 1987, Cell48:193-203; Lelkes and Pollard, 1987, J. Biol. Chem. 262:15496-14505).

In order to exclude a possible general enhancement of C6 cell spreadingon nonpermissive substrates, we tested metalloprotease-dependent C6 cellspreading on two other substrates in addition to PLYS and CNS myelin(FIG. 22): PNS myelin and glass. PNS myelin was chosen as a control forthe general properties of a myelin membrane fraction (e.g., high contentof lipids), and glass was chosen because of its well known bad substratequalities. Half maximal inhibition of spreading on CNS myelin wasobtained with 200 μM 1,10-phenanthroline. On PLYS, glass, and PNS myelin(FIG. 22), 1,10-phenanthroline did not impair C6 cell spreading atconcentrations up to 0.5 mM (FIG. 22).

Absorption of CNS myelin with a monoclonal antibody (IN-1) raisedagainst CNS myelin inhibitory components (see Section 8.1.3., supra)largely reversed 1,10-phenanthroline dependent inhibition of C6 cellspreading on CNS myelin liposomes (Table XI). IN-1 also almostcompletely neutralized the inhibitory substrate property of CNS myelinprotein liposomes for 3T3 cells (Table XI).

                  TABLE XI                                                        ______________________________________                                        INHIBITION OF C6 CELL SPREADING BY                                            1,10-PHENANTHROLINE ON CNS MYELIN                                             IS NEUTRALIZED BY ANTIBODY IN-1*                                                                             % inhibition                                            1,10-Phenan-                                                                          spreading value on:                                                                         on CNS                                         Cells                                                                              Antibody  throline  CNS lipos.                                                                            PLYS  lipos.                                 ______________________________________                                        3T3  --        0         1.11    2.00  45                                     3T3  IN-1      0         2.03    2.26  10                                     3T3  mouse IgM 0         1.16    2.18  47                                     C6   --        0         2.48    2.52   2                                     C6   --        0.3 mM    1.35    2.49  46                                     C6   IN-1      0         2.46    2.48   1                                     C6   IN-1      0.3 mM    2.25    2.54  11                                     C6   mouse IgM 0         2.36    2.42   2                                     C6   mouse IgM 0.3 mM    1.41    2.39  41                                     ______________________________________                                         *CNS myelin protein liposomes were used as substrates, and were               preadsorbed with monoclonal antibody IN1 against the myelin inhibitory        substrate constituents (see Section 8.1.4), or with mouse IgM. Spreading      was calculated after 150 minutes and is expressed as μm.sup.2 10.sup.3     % Inhibition relates to spreading values on PLYS.                        

These results indicate that the metalloprotease(s) plays an importantrole for overcoming of CNS myelin inhibitory substrates byneutralization Of IN-1 inhibitory properties.

9.2.4. A C6 PLASMA MEMBRANE ASSOCIATED ACTIVITY NEUTRALIZES THEINHIBITORY SUBSTRATE PROPERTY OF CNS MYELIN

CNS myelin-coated culture wells were incubated with C6 conditionedmedium or C6 plasma membranes, and subsequently tested for theirinhibitory substrate property with spreading of 3T3 cells. We found thatC6 plasma membranes contained an activity which strongly reduced CNSmyelin inhibitory activity (FIGS. 23A-D, Table XII). The same treatmentalso decreased the inhibitory effect of CNS myelin protein liposomes orSDS-PAGE-purified, reconstituted 35 kD and 250 kD inhibitory components.The decrease in CNS myelin inhibitory activity for 3T3 cell adhesion andspreading was quantitified by measuring spreading values and DNAsynthesis (Table XII).

                  TABLE XII                                                       ______________________________________                                        C6 PLASMA MEMBRANES REDUCE CNS MYELIN                                         INHIBITORY SUBSTRATE PROPERTY FOR 3T3 CELLS*                                                     3T3 Cell .sup.3 H-Thymidine                                                   Spreading                                                                              Incorporation                                     Substrates         (%)      (%)                                               ______________________________________                                        PLYS               100      100                                               CNS myelin         15       30                                                CNS myelin, C6 PM  52       83                                                CNS myelin, C6 PM, phen. treated                                                                 17       50                                                CNS myelin, C6 PM, EDTA treated                                                                  13       nd                                                ______________________________________                                         *3T3 cells were plated on PLYS or CNS myelin. Spreading was assessed afte     150 minutes CNS myelin was preincubated with a C6 cell plasma membrane        fraction (C6 PM) in the absence or presence of metalloprotease inhibitors     as indicated.                                                                 .sup.3 Hthymidine was added when 3T3 cells were plated, and incorporation     was determined after 20 hours                                                 nd: not determined.                                                      

1,10-phenanthroline, EDTA, and the dipeptide cbz-gly-phe-NH₂ completelyblocked the C6 plasma membrane effect. Trasylol, leupeptine andpepstatine did not inhibit this effect. C6 conditioned medium used assuch, or 10-times concentrated, did not contain any degradative activityable to neturalize CNS myelin inhibitory substrate properties.

9.2.5. INHIBITORS OF METALLOPROTEASES IMPAIR C6 CELL SPREADING ON CNSWHITE MATTER AND C6 INFILTRATION OF CNS EXPLANTS

In order to investigate the relevance of the C6 plasma membranemetalloprotease activity not only for C6 cell attachment and spreading,but also for C6 cell migration and infiltration, C6 cells were plated oncerebellar frozen sections or added to optic nerve explants in thepresence of two metalloprotease inhibitors (1,10-phenanthroline andcbz-tyr-tyr). Parallel cultures contained inhibitors for the three otherclasses of proteases (leupeptine, pepstatine or trasylol), or a controldipeptide (cbz-ala-phe).

The presence of 1,10-phenanthroline at different concentrations (50,100, 200 and 300 μM), or the dipeptide cbz-tyr-tyr (100 μM) dramaticallychanged the distribution and behavior of C6 cells on the white matterareas when cerebellar frozen sections were used as culture substrates(FIG. 23). C6 cells also adhered in large numbers and spread extensivelyon the grey matter (FIGS. 23A-D).

Rat optic nerves were injected with 4 μl of 3 mM solutions of eithercbz-ala-phe or cbz-tyr-tyr. Cells were incubated with medium containing0.5 mM peptide. In the outer chamber, where no cells were present, thepeptide concentration was 1 mM. After 14 days, the immigration of C6cells into the explants differed greatly (FIGS. 24A-B).cbz-ala-phe-injected nerves contained more cells, and C6 cellinfiltration was not affected, as compared to explants injected withculture medium only. On the other hand, cbz-tyr-tyr inhibited C6 cellinfiltration in all the 8 nerves examined (2 experiments). C6 cells werefound mainly at the cut end of these nerve explants, and deepinfiltration, which occurred massively in control explants, was stronglyreduced by cbz-tyr-tyr.

9.3. DISCUSSION

The present results demonstrate that C6 glioblastoma cells, in contrastto neurons, fibroblasts and B16 melanoma cells, were not impaired intheir migration into optic nerve explants or in attachment and spreadingon CNS white matter, isolated CNS myelin, or living oligodendrocytes.The fact that the behavior of C6 cells differed characteristically fromthat of several cell types in all the assay systems studied suggestscommon underlying cell biological mechanisms, both for C6 spreading onan inhibitory substrate as well as for C6 mobility in an environment(optic nerve) which does not allow fibroblasts, Schwann cell or melanomacell migration nor does it allow ingrowth of regenerating nerve fibers.This behavior of C6 cells was not due to "insensitivity" to theinhibitory components, since C6 cell motility was drastically inhibitedon CNS myelin or white matter in the presence of specific proteaseblockers, and this effect was reversed by selective neutralization ofmyelin-associated inhibitory proteins with a monoclonal antibody (IN-1).

Inactivation of myelin-associated inhibitory constituents occurred byliving C6 cells as well as by C6 plasma membranes. Our experiments witha number of protease blockers with different known specificities showedthat this C6 associated activity belongs to the metalloenzyme family.The close parallelism observed between prevention of C6 cell spreadingon CNS myelin and prevention of inactivation of myelin-associatedinhibitory proteins strongly suggests that modification of theinhibitory substrate components by a metalloprotease could be themechanism which enables C6 cells to spread on myelin, on white matter,and to infiltrate optic nerve explants.

Metalloproteases form an increasingly numerous group, the members ofwhich differ in their sensitivity to various blockers. The most generalblocker is 1,10-phenanthroline which impaired C6 cell spreading on CNSmyelin up to 67%, whereas most inhibitors of the other classes ofproteases had no detectable effects. In the early (90 minutes) but notthe later (300 minutes) phases of C6 cell spreading on myelin, an effectof trypsin-like serine-protease inhibitors was also observed. The effectof 1,10-phenanthroline was dose-dependent, with an IC₅₀ of 200 μM. Thiseffect was specific for CNS myelin as a substrate, since normal, rapidspreading of C6 cells was observed on other substrates such as CNS greymatter, PNS myelin, glass or PLYS in the presence of1,10-phenanthroline. Other known metalloprotease blockers like bestatine(inhibitor of aminopeptidases; Umezawa, et al., 1976, J. Antibiot.29:857-859), phosphoramidone (inhibitor of thermolysin-likemetalloproteases; Komiyama, et al., 1975, Biochem. Biophys. Res. Commun.65:352-357) and TIMP (inhibitor of ECM degrading metalloproteases;Cawston, et al., 1981, 195:159-165) did not lead to inhibition of C6cell spreading on CNS myelin. Since metalloproteases generally hydrolyzepeptide bonds followed by large aliphatic or neutral aromatic aminoacids, we tested the effect of dipeptide substrate analogues containingsuch residues. Cbz-gly-phe-NH₂ (1 mM) or cbz-tyr-tyr (0.3 mM) inhibitedC6 cell spreading specifically on CNS myelin. Cbz-gly-phe-NH₂ was foundto inhibit other 1,10-phenanthroline sensitive enzyme activities withrelative high specificity (Almenoff and Orlowski, 1983, Biochemistry22:590-599; Baxter, et al., 1983, Proc. Natl. Acad. Sci. U.S.A.80:4174-4178; Couch and Strittmatter, 1983, Cell 32:257-265; Chen, J. M.and Chen, W. T., 1987, Cell 48:193-203; Lelkes and Pollard, 1987, J.Biol. Chem. 262:15496-14505).

Inactivity of C6-conditioned medium and cell fractionation experimentsdemonstrated that the myelin-directed proteolytic actiity is associatedwith C6 plasma membranes. The isolation and characterization of a plasmamembrane-bound metalloprotease (endopeptidase 24.11, enkephalinase),which is also blocked by 1,10-phenanthroline but not by TIMP, wasreported by Almenoff and Orlowski (1983, supra). However, themetalloprotease described herein is probably not an enkephalinase, sincecarboxymethyl-phe-leu, a peptide with high affinity for enkephalinase(Fournie-Zaluski, et al., 1983, J. Med. Chem. 26:60-65), did not affectC6 spreading on myelin. A metalloprotease expressed by Rous sarcomavirus transformed chick embryo fibroblasts and localized at adhesionsites and on "invadopodia" was described by Chen, and Chen, 1987, supra.This enzyme is also inhibited by 1,10-phenanthroline andcbz-gly-phe-NH₂, but not by phosphoramidon, as is the metalloproteasedescribed here. However, unlike the enzyme of Chen and Chen, we couldnot detect any fibronectin degradative activity on C6 cells.

The highly metastatic B16 mouse melanoma cells were tested in all theassays used with C6 cells. Interestingly, B16 cells did not migrate intooptic nerve explants, but responded to the myelin-associated inhibitorsin a way very similar to 3T3 cells or neurons. In line with this invitro behavior, B16 cells, upon intraventricular injection, form mainlymeningiomas or intraventricular tumors without significant infiltrationof the brain parenchyma. Thus. the mechanisms providing metastaticbehavior to B16 cells in the periphery are different from thoseconferring high mobility to C6 cells in the CNS tissue.

Inhibition of C6-associated metalloprotease not only inhibited C6spreading on CNS myelin, but also abolished C6 cell attachment,spreading, and migration on CNS white matter, and the dipeptide,cbz-tyr-tyr strongly impaired the migration of C6 cells into optic nerveexplants. This metalloprotease activity(ies) may, therefore, becrucially involved in the infiltrative behavior of C6 glioblastoma cellsin CNS tissue, also in vivo.

10. LONG DISTANCE TRACT REGENERATION IN THE LESIONED SPINAL CORD OF RATSBY A MONOCLONAL ANTIBODY AGAINST MYELIN-ASSOCIATED NEURITE GROWTHINHIBITORS

The monoclonal antibody IN-1, which neutralizes the inhibitory substrateeffect of the 35 kD and 250 kD myelin-associated proteins and of CNStissue explants (Caroni and Schwab, 1988, Neuron 1:85-96), was appliedto young rats intracerebrally by implanting antibody producing tumorsinto the neocortex. Complete transections of the cortico-spinalcomponent of the pyramidal tract (CST) at 2-4 weeks of age was followedby massive sprouting around the lesion, and, in IN-1 treated rats, byelongation of fine axons and fascicles up to 8-11 mm distal to thelesion within 2 weeks. In control rats the maximal distance of observedelongation rarely exceeded 1 mm. These results demonstrate the inducedregeneration capacity of a major motor CNS tract within differentiatedCNS tissue, and point to the clinical importance of CNS neurite growthinhibitors and their antagonists.

10.1. MATERIALS AND METHODS 10.1.1. PRE-OPERATIVE PREPARATION OF ANIMALSINCLUDING IMPLANTATION OF HYBRIDOMA CELLS

Young Lewis rats (P2-11) were injected unilaterally under etheranesthesia into the dorsal frontal cortex with 1 Mio. hybridoma cells in1 or 2 μl. Control rats were injected with the same number of cells of ahybridoma line producing antibodies against horseradish peroxidase(HRP). Non-injected controls were also used. Hybridoma cells: IN-1secreting cells were obtained by fusion of P3U myeloma cells with spleencells of a BALB/c mouse immunized against the PAGE-purified 250 kDinhibitory protein fraction from rat spinal cord myelin as described byCaroni and Schwab (1988, Neuron 1:85-96); anti-HRP secreting cells wereobtained by Dr. P. Streit, Zurich, according to the protocol ofSemenenko et al. (1985 Histochem. 83:405-408) using the same myelomaline (P3U) as for IN-1. In all hybridoma-injected rats, tumors formedwithin a few days as solid, well delineated tumors often spanning theentire thickness of the neocortex and contacting the lateral ventricle(FIGS. 26A-B). Cyclosporin A injections (15 μg/g body weight, 2injections at 3 day intervals) helped to prevent tumor resorption whichotherwise occurred after 2-3 weeks. Massive production of antibodiescould be detected by staining brain sections with anti-mouse Ig-FITC(FITC-conjugated immunoglobulin) (FIG. 26B), and by the presence of In-1antibodies in the serum (data not shown).

10.1.2. PROCEDURE FOR PERFORMING SPINAL CORD LESION

Spinal cord lesions were placed at 2-4 weeks of age (Table III, infra)at the thoracic level T₅₋₇ by slightly separating two vertebrae andtransecting the dorsal two thirds of the spinal cord with iridectomyscissors. The lesion completely transsected the CSTs of both sidesincluding the lateral projections into the dorsal gray matter, and alsothe central canal. Ventral and lateral white matter remainedundisturbed, allowing the rats a seemingly normal behavior. Lesions weredone at 15-29 days of age, i.e. 5-20 days after termination of axongrowth in the CST (Table XIII). A U-shaped stainless steel wire was thenimplanted into the lesion site in order to assure complete transectionof both CSTs and to mark the lesion site. (The wire was removed prior toembedding the fixed spinal cords for sectioning).

                  TABLE XIII                                                      ______________________________________                                        REGENERATION OF CORTICO-SPINAL TRACT                                          AXONS AFTER MID-THORACIC LESIONS IN                                           CONTROL AND ANTIBODY IN-1 TREATED RATS                                        Tumor-  Day of    Survival                                                                              Max. distance of regenerated                        type    lesion    time    CST axons caudal to lesion                          ______________________________________                                        none    P 14      19 d.   0.1                                                                           0.2                                                                           0.2                                                                           0.5                                                 none    P 22      14 d.   0.4                                                                           0.2                                                 none    P 22      11 d.   0.7                                                                           0.6                                                 αHRP                                                                            P 15      14 d.   0.4                                                                           1.0                                                                           1.8                                                                           2.6                                                 αHRP                                                                            P 18      16 d.   0.1                                                                           0.2                                                                           0.2                                                                           0.3                                                                           0.4                                                                           0.5                                                                           0.8                                                 IN-1    P 14      16 d.   2                                                                             >8*                                                                           11                                                  IN-1    P 15      15 d.   4                                                                             4.5                                                                           >5*                                                                           >5                                                                            >5                                                  IN-1    P 18      18 d.   2.5                                                 IN-1    P 19      14 d.   7.7                                                                           7.8                                                 IN-1    P 28      14 d.   >4                                                                            >4                                                  IN-1    P 29      27 d.   >2.5                                                ______________________________________                                         Methods as described in FIG. 27. Only rats with regenerative CST sprouts      caudal to the lesion were included in this analysis. Distances of             regenerating fibers are measured from the caudal edge of the lesion           caverns.                                                                      *Minimal distance as regenerating fibers reach the caudal end of the          tissue block.                                                            

10.1.3. POST-LESION EVALUATION

After survival times of 14-28 days (Table XIII), the frontal andparietal cortex contralateral to the tumor was injected with a 5%solution of WGA-HRP (1 μl). Twenty-four hours later, rats were perfusedthrough the heart with 1.25% glutaraldehyde and 1% formaldehyde in 0.1 Mphosphate buffer for 10 minutes. The dissected spinal cords (10-15 mm)were postfixed in the same fixative for 1 hour, extensively washed, andembeded for cryostat sectioning. Complete longitudinal section serieswere mounted on gelatin-coated slides, and reacted for HRP using TMB asa substrate (Mesulam, 1978, J. Histochem. & Cytochem. 26:106-117).Sections were viewed under dark-field illumination in polarized light.Only rats with complete bilateral CST lesions and with sprouts appearingon the caudal side of the lesion were evaluated.

10.2. RESULTS: REGENERATION OF CORTICOSPINAL TRACT (CST) FIBERS OVERLONG DISTANCES IN RATS BEARING IN-1 SECRETING TUMORS

Two weeks after the lesions, at or beyond 1 month of age, the CST waslabeled by anterograde transport of WGA-HRP from the frontal andparietal cortex. The histological examination of the lesion site intransverse and longitudinal sections showed a very similar picture inall animals: usually several small caverns were present and communicatedwith the central canal, a feature which probably greatly enhanced thelocal access and penetration of the antibodies carried down by thecerebrospinal fluid. The tissue was locally altered, but no dense glialscars were present. Labeled CST fibers approached the lesion as a denseand compact bundle from which massive sprouting occurred 0.5-1 mmproximal to the lesion. In most animals, controls or IN-1-injected,fiber plexus and bundles were seen in and across the lesion area, mostoften circumventing the lesion caverns ventrally or laterally, butrarely also growing through tissue bridges that had reformed in the wiretract. Fibers leaving the lesion site and travelling in a caudaldirection could frequently be observed. In animals without tumors and inrats with anti-HRP-producing tumors, the travelling distances measuredon longitudinal sections from the distal edge of the lesion were in mostinstances below 1 mm (Table XIII, FIGS. 27,28A-G). Even relatively thickfascicles seemed to end abruptly. Very much in contrast, animals bearingIN-1 secreting tumors consistently showed labelled fascicles and fibersat much longer distances caudal to the lesion (FIGS. 27,28A-G). 2.5-5 mmwere measured in most animals, 8 and 11 mm were seen in 2 rats (TableXIII). Anatomically, these long distance regenerating CST fibers weremost often found close to or in the original CST location, with somefibers also in the gray matter and a few fibers in more dorsal regionscorresponding to the sensory tracts.

10.3. DISCUSSION

In the rat, the CST is known to grow down the spinal cord during thefirst 10 postnatal days, the last axons being added at P9-P10 (Joostenet al., 1987, Dev. Brain Res. 36:121-139; Schreyer and Jones, 1988 Dev.Brain Res. 38:103-119). Lesions of the tract up to P4-P5 lead to acircumvention of the lesion site and to long-distance, often ectopicgrowth of CST fibers (Schreyer and Jones, 1983, Neurosci. 9:31-40;Bernstein and Stelzner, 1983, J. Comp. Neurol. 221:382-400). Noregeneration in the CST has been seen after P6. A very similar lesionresponse has been observed in hamster and cat (Kalil and Reh, 1982, J.Comp. Neurol. 211:265-275; Tolbert and Der, 1987, J. Comp. Neurol.260:299-311). For the cat it was demonstrated that these fibers aremostly late-arriving, newly-growing, rather than regenerating axons(Tolbert and Der, 1987, J. Comp. Neurol. 211:265-275). The presentresults demonstrate that at least a small proportion of CST neurites at2-3 weeks of age can be induced to regenerate and elongate over longdistances inside the spinal cord. The maximal speed of elongation is inthe range of 0.5-1 mm/day.

Differentiated CNS tissue of mammals is a nonpermissive substrate forneurite growth beyond a sprouting distance of between 0.2-1 mm (Cajal,1959, in "Degeneration and Regeneration of the Nervous System," ed.Hafner, New York, p. 1928; David, 1981, Science 214:931-933; andVidal-Sanz et al., 1987, J. Neurosci. 7:2894-2909). This property isexpressed (far more by CNS white matter than CNS gray matter, as shownby culture experiments and by transplantation studies (Schwab andThoenen, 1985, J. Neurosci. 5:2415-2423; Carbonetto et al., 1987, J.Neurosci. 7:610-620; Savio and Schwab, 1989, in press). Transplantationsof fetal adrenergic or serotoninergic neurons of defined fetal ages intoadult spinal cords or hippocampus represent up to now the only otherexperiments where elongation of axons in adult CNS tissue was observedat an anatomical level (Nornes et al., 1983 Cell Tissues Res. 230:15-35;Foster et al., 1985 Exp. Brain Res. 60:427-444 and Bjorklund et al.,1979 Brain Res. 170:409-426). These elongating axons were almostexclusively localized to gray matter areas.

Two oligodendrocyte- and myelin-associated membrane proteins, NI-35 (35kD) and NI-250 (250 kD), with potent inhibitory effects on neuritegrowth, were identified by in vitro and biochemical studies (Schwab andCaroni, 1988 J. Neurosci. 8:2381-2393; Caroni and Schwab, 1988, J. Cell.Biol. 106:1281-1288). Monoclonal antibody IN-1, which neutralizes theactivity of these constituents in various in vitro systems includingadult rat optic nerve explants (Caroni and Schwab, 1988, Neuron1:85-96), is shown here to lead to true regeneration of cortico-spinalaxons in young rats over distances of up to 5-11 mm distal to a spinalcord lesion within 2 weeks. The continuous supply of high levels ofantibodies via the cerebrospinal fluid by an antibody-secreting tumor inthe cortex, and the local conditions of the lesion probably helped thepenetration of the antibodies into the tissue. The absence of axonelongation distal to the lesion in spite of massive sprouting around thelesion site in animals bearing control antibody tumors confirms thespecificity of the effect observed. These results clearly demonstratethe ability of antibodies directed toward the myelin-associated neuritegrowth inhibitor protein to induce neuron fiber regeneration over longdistances, as well as the crucial role of the myelin-associated neuritegrowth inhibitors for the absence of regeneration of lesioned CNS fibertracts observed under normal conditions.

11. DEPOSIT OF MICROORGANISMS

The following hybridomas, producing the indicated monoclonal antibodies,have been deposited Oct. 28, 1998 with the European Collection of AnimalCell Cultures (ECACC), PHLS Centre for Applied Microbiology andResearch, Porton Down, Salisbury, Wiltshire, United Kingdom, and havebeen assigned the listed accession numbers.

    ______________________________________                                        Hybridoma     Antibody Accession Number                                       ______________________________________                                        Cell line IN-1                                                                              IN-1     88102801                                               Cell line IN-2                                                                              IN-2     88102802                                               ______________________________________                                    

The present invention is not be limited in scope by the cell linesdeposited or the embodiments disclosed in the examples which areintended as illiustrations of a few aspects of the invention and anyembodiment which are functionally equivalent are within the scope ofthis invention. Indeed, various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art and are intended to fall within the scope ofthe appended claims.

What is claimed is:
 1. A method for treating a patient with a malignant tumor that originates in the central nervous system comprising administering a therapeutically effective amount of a metalloprotease inhibitor to the patient.
 2. The method according to claim 1 in which the malignant tumor is a glioblastoma.
 3. The method according to claim 1 in which the metalloprotease inhibitor is selected from the group consisting of 1,10 phenanthroline, ethylenediamine tetraacetate, ethyleneglycol-bis (β-aminoethyl ether) N, N, N'-N'-tetraacetate, carbobenzoxy-tyrosine-tyrosine, carbobenzoxy-glycine-phenylalanine-amide, carbobenzoxy-phenylalanine-tyrosine-amide, carbobenzoxy-phenylalanine-phenylalanine-amide, and carbobenzoxy-glycine-phenylalanine-phenylalanine-amide.
 4. The method according to claim 1 in which the patient is a human.
 5. A method for treating a patient with a malignant tumor that originates in the central nervous system comprising administering a therapeutically effective amount to the patient of a compound which inhibits the metalloprotease activity of a neurite growth regulatory factor, said factor consisting of a molecule isolated from glioblastoma cells and characterized by the following properties:(a) metalloprotease activity; and (b) neutralizes the nonpermissive substrate property of the CNS myelin of a higher vertebrate, in which the neutralization is detected by observing the ability of the CNS myelin in the presence of the factor to support neurite outgrowth or fibroblast spreading in vitro.
 6. The method according to claim 5 in which the compound inhibits C6 cell spreading and migration on CNS myelin of a higher vertebrate wherein the inhibition can be reversed by a substance which neutralizes the non-permissive substrate property of the CNS myelin of a higher vertebrate.
 7. The method according to claim 6 in which the inhibition of C6 cell spreading and migration by the compound can be reversed by monoclonal antibody IN-1, as produced by cell line IN-1, as deposited with the ECACC and assigned accession number
 88102801. 8. The method according to claim 5 in which the tumor is a glioblastoma.
 9. The method according to claim 5 in which the compound is selected from the group consisting of 1,10-phenanthroline, ethylenediamine tetraacetate, ethyleneglycol-bis (β-aminoethyl ether) N, N, N'-N'-tetraacetate, carbobenzoxy-tyrosine-tyrosine, carbobenzoxy-glycine-phenylalanine-amide, and carbobenzoxy-phenylalanine-tyrosine-amide, carbobenzoxy-phenylalanine-phenylalanine-amide, and carbobenzoxy-glycine-phenylalanine-phenylalanine-amide.
 10. The method according to claim 5 in which the patient is a human. 